CN116234907A

CN116234907A - RNAI constructs for inhibiting HSD17B13 expression and methods of use thereof

Info

Publication number: CN116234907A
Application number: CN202180060123.2A
Authority: CN
Inventors: D·C·H·林; M·奥尔曼; J·K·默里; B·J·赫伯里希; A·达斯; P·柯林斯; O·霍曼
Original assignee: Amgen Inc
Current assignee: Amgen Inc
Priority date: 2020-06-01
Filing date: 2021-06-03
Publication date: 2023-06-06
Also published as: KR20240004092A; JP2023528608A; WO2021247885A3; CL2022003369A1; US20230279399A1; EP4158026A2; WO2021247885A2; MX2022015169A; IL298697A; BR112022024501A2; CA3184345A1; AU2021284377A1

Abstract

The present invention relates to RNAi constructs for reducing expression of the HSD17B13 gene. Methods of using such RNAi constructs to treat or prevent liver disease, non-alcoholic fatty liver disease (NAFLD) are also described.

Description

RNAI constructs for inhibiting HSD17B13 expression and methods of use thereof

Technical Field

The present invention relates to compositions and methods for modulating liver expression of 17 beta-hydroxysteroid dehydrogenase type 13 (HSD 17B 13), in particular, the present invention relates to nucleic acid-based therapeutics for reducing HSD17B13 expression via RNA interference, and methods of using such nucleic acid-based therapeutics for treating or preventing liver disease, such as non-alcoholic fatty liver disease (NAFLD).

Background

Non-alcoholic fatty Liver disease (NAFLD) constitutes a series of Liver lesions, the most common chronic Liver disease in the world, with a double prevalence in the last 20 years and is now estimated to affect about 20% of the world population (Sattar et al (2014) BMJ [ J. British medical journal ]349: g4596; loomba and Sanyal (2013) Nature Reviews Gastroenterology & hepatology [ natural reviews, gastroenterology and hepatology ]10 (11): 686-690; kim and Kim (2017) Clin Gastroenterol Hepatol [ clinical gastroenterology and hepatology ]15 (4): 474-485; petta et al (2016) Dig Liver Dis [ gastroenteropathy and hepatopathy ]48 (3): 333-342). NAFLD begins triglyceride accumulation in the liver and is defined as the presence of cytoplasmic lipid droplets in more than 5% of hepatocytes in an individual 1) without a significant history of alcohol consumption and 2) in which diagnosis of other types of liver disease has been excluded (Zhu et al (2016) World JGastroenterol [ J.WHO.J.22 (36): 8226-33; rinella (2015) JAMA [ journal of the American medical society ]313 (22): 2263-73; yki-Jarvonn (2016) diabetes 59 (6): 1104-11). In some individuals, the accumulation of ectopic fat in the liver, known as steatosis, causes inflammation and hepatocyte damage, leading to more advanced disease, known as nonalcoholic steatohepatitis (NASH) (Rinella, supra). By 2015, 7500 ten thousand to 1 hundred million americans are expected to have NAFLD; NASH accounts for about 10% -30% of NAFLD diagnosis (Rinella, supra; younossi et al (2016) Hepatology ]64 (5): 1577-1586).

17 beta-hydroxysteroid dehydrogenase type 13 (HSD 17B 13), also known as 17 beta-HSD 13, is a member of the 17 beta-hydroxysteroid dehydrogenase (HSD 17B) family, which includes the family of enzymes that catalyze the conversion between 17-keto-and 17-hydroxysteroids (Su et al (2019) Molecular and Cellular Endocrinology [ molecular and cytoendocrinology ]; 489:119-125). HSD17B13 was originally designated as SCDR9 and was first cloned in 2007 from a human liver cDNA library (Liu et al (2007) Acta Biochim [ journal of biochemistry ] 54:213-218). In 2008, horiguchi identified HSD17B13 as a novel LD-related protein whose expression was primarily restricted to the liver (Horiguchi et al (2008) Biochem Biophys Res Commun [ communication of biochemical and biophysical studies ] 370:235-238). Liver overexpression of HSD17B13 promotes lipid accumulation in the liver. HSD17B13 expression was significantly upregulated in patients and mice with non-alcoholic fatty liver disease (NAFLD) (Su et al (2014) PNAS 111:11437-11442). Thus, new therapies targeting HSD17B13 represent a new approach to reduce HSD17B13 levels and treat liver disease (e.g., non-alcoholic fatty liver disease).

Disclosure of Invention

The present invention is based in part on the design and generation of RNAi constructs that target HSD17B13 mRNA and reduce HSD17B13 expression in liver cells. Sequence-specific inhibition of HSD17B13 expression may be useful in the treatment or prevention of conditions associated with HSD17B13 expression, such as liver-related diseases, such as, for example, simple fatty liver (steatosis), nonalcoholic steatohepatitis (NASH), cirrhosis (irreversible, advanced scarring of the liver), or HSD17B 13-related obesity. Thus, in one embodiment, the invention provides an RNAi construct comprising a sense strand and an antisense strand, wherein the antisense strand comprises a region having a sequence complementary to the HSD17B13 mRNA sequence. In certain embodiments, the antisense strand comprises a region of at least 15 contiguous nucleotides having an antisense sequence from table 1 or table 2.

In some embodiments, the sense strand of the RNAi constructs described herein comprises a sequence sufficiently complementary to the sequence of the antisense strand to form a duplex region of about 15 to about 30 base pairs in length. In these and other embodiments, the sense strand and the antisense strand are each about 15 to about 30 nucleotides in length. In some embodiments, the RNAi construct comprises at least one blunt end. In other embodiments, the RNAi construct comprises at least one nucleotide overhang. Such nucleotide overhangs may comprise at least 1 to 6 unpaired nucleotides and may be located at the 3' end of the sense strand, the 3' end of the antisense strand, or the 3' end of both the sense and antisense strands. In certain embodiments, the RNAi construct comprises two overhangs of unpaired nucleotides at the 3 'end of the sense strand and the 3' end of the antisense strand. In other embodiments, the RNAi construct comprises two unpaired nucleotide overhangs at the 3' end of the antisense strand and a blunt end at the 3' end of the sense strand/5 ' end of the antisense strand.

RNAi constructs of the invention may comprise one or more modified nucleotides, including nucleotides modified to the ribose ring, nucleobase, or phosphodiester backbone. In some embodiments, the RNAi construct comprises one or more 2' -modified nucleotides. Such 2 '-modified nucleotides may include 2' -fluoro modified nucleotides, 2 '-O-methyl modified nucleotides, 2' -deoxy modified nucleotides, 2 '-O-methoxyethyl modified nucleotides, 2' -O-allyl modified nucleotides, bicyclic Nucleic Acids (BNA), diol nucleic acids (GNA), reverse bases (e.g., reverse adenosine), or combinations thereof. In a particular embodiment, the RNAi construct comprises one or more 2 '-fluoro modified nucleotides, 2' -O-methyl modified nucleotides, or a combination thereof. In some embodiments, all nucleotides in the sense and antisense strands of the RNAi construct are modified nucleotides.

In some embodiments, the RNAi construct comprises at least one backbone modification, e.g., a modified nucleotide or internucleoside linkage. In certain embodiments, RNAi constructs described herein comprise at least one phosphorothioate internucleotide linkage. In particular embodiments, phosphorothioate internucleotide linkages may be located at the 3 'or 5' end of the sense strand and/or antisense strand.

In some embodiments, the antisense strand and/or sense strand of an RNAi construct of the invention can comprise or consist of sequences from the antisense and sense sequences listed in table 1 or 2. In certain embodiments, the RNAi construct can be any one of the duplex compounds listed in any one of tables 1-2.

Detailed Description

The present invention is directed to compositions and methods for modulating the expression of a 17 beta-hydroxysteroid dehydrogenase type 13 (HSD 17B 13) gene. In some embodiments, the gene may be within a cell or subject, such as a mammal (e.g., a human). In some embodiments, the compositions of the invention comprise RNAi constructs that target HSD17B13 mRNA and reduce HSD17B13 expression in a cell or mammal. Such RNAi constructs can be used to treat or prevent various forms of liver-related diseases, such as, for example, simple fatty liver (steatosis), nonalcoholic steatohepatitis (NASH), cirrhosis (irreversible, advanced scarring of the liver), or HSD17B 13-related obesity.

RNA interference (RNAi) is a process by which exogenous RNA is introduced into a cell, resulting in specific degradation of mRNA encoding a target protein, and thus in reduced protein expression. Advances in both RNAi technology and liver delivery and the ever-increasing positive results with other RNAi-based therapies suggest that RNAi is a powerful means of therapeutic treatment of NAFLD by direct targeting of HSD17B 13.

As used herein, the term "RNAi construct" refers to an agent comprising an RNA molecule capable of down-regulating the expression of a target gene (e.g., HSD17B 13) via an RNA interference mechanism upon introduction into a cell. RNA interference is the process by which a nucleic acid molecule induces cleavage and degradation of a target RNA molecule (e.g., via a messenger RNA or mRNA molecule) in a sequence-specific manner (e.g., via an RNA-induced silencing complex (RISC) pathway). In some embodiments, the RNAi construct comprises a double-stranded RNA molecule comprising two antiparallel strands of consecutive nucleotides, which are sufficiently complementary to each other to hybridize to form a duplex region. "hybridization" refers to the pairing of complementary polynucleotides, typically via hydrogen bonding between complementary bases in two polynucleotides (e.g., watson-Crick, hu Gesi butane (Hoogsteen), or reverse Hu Gesi Ding Qingjian). The strand comprising a region having a sequence substantially complementary to a target sequence (e.g., target mRNA) is referred to as the "antisense strand". "sense strand" refers to a strand that includes a region that is substantially complementary to a region of an antisense strand. In some embodiments, the sense strand may comprise a region having substantially the same sequence as the target sequence.

In some embodiments, the invention is directed to an RNAi construct to HSD17B 13. In some embodiments, the invention includes RNAi constructs comprising any of the sequences found in table 1 or table 2.

The double-stranded RNA molecule may include chemical modifications to the ribonucleotides, including modifications to the ribose, base, or backbone components of the ribonucleotides, such as those described herein or known in the art. For the purposes of this disclosure, the term "double stranded RNA" encompasses any such modification used in double stranded RNA molecules (e.g., siRNA, shRNA, etc.).

As used herein, a first sequence is "complementary" to a second sequence if a polynucleotide comprising the first sequence can hybridize to a polynucleotide comprising the second sequence to form a duplex region under certain conditions (e.g., physiological conditions). Other such conditions may include moderate or stringent hybridization conditions known to those of ordinary skill in the art. A first sequence is considered to be fully complementary (100% complementary) to a second sequence if the polynucleotide comprising the first sequence base pairs with the polynucleotide comprising the second sequence without any mismatches over the entire length of one or both nucleotide sequences. A sequence is "substantially complementary" to a target sequence if it is at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% complementary to the target sequence. The percent complementarity may be calculated by dividing the number of bases in the first sequence that are complementary to bases at corresponding positions in the second or target sequence by the total length of the first sequence. A sequence is also said to be substantially complementary to another sequence if there are no more than 5, 4, 3, 2 or 1 mismatches in a 30 base pair duplex region when the two sequences hybridize. In general, if any nucleotide overhangs as defined herein are present, the sequence of these overhangs is not considered in determining the degree of complementarity between the two sequences. For example, a sense strand of 21 nucleotides in length and an antisense strand of 21 nucleotides in length hybridize to form a 19 base pair duplex region having a 2 nucleotide overhang at the 3' end of each strand, which will be considered fully complementary, as that term is used herein.

In some embodiments, the region of the antisense strand comprises a sequence that is fully complementary to a region of the target RNA sequence (e.g., HSD17B13 mRNA). In such embodiments, the sense strand may comprise a sequence that is fully complementary to the sequence of the antisense strand. In other such embodiments, the sense strand may comprise a sequence that is substantially complementary to the sequence of the antisense strand, e.g., 1, 2, 3, 4, or 5 mismatches in the duplex region formed by the sense strand and the antisense strand. In certain embodiments, it is preferred that any mismatches occur within the terminal region (e.g., within 6, 5, 4, 3, 2, or 1 nucleotides of the 5 'and/or 3' ends of the strand). In one embodiment, any mismatch in the duplex region formed by the sense strand and the antisense strand occurs within 6, 5, 4, 3, 2, or 1 nucleotides of the 5' end of the antisense strand.

In certain embodiments, the sense strand and the antisense strand of the double-stranded RNA can be two separate molecules that hybridize to form a duplex region, but are otherwise not joined. Such double stranded RNA molecules formed from two separate strands are referred to as "small interfering RNAs" or "short interfering RNAs" (siRNAs). Thus, in some embodiments, RNAi constructs of the invention comprise siRNA.

When the two substantially complementary strands of a dsRNA consist of separate RNA molecules, those molecules need not be, but can be, covalently linked. If two strands are covalently joined by means other than an uninterrupted nucleotide chain between the 3 'end of one strand and the 5' end of the corresponding other strand, the joined structure is referred to as a "linker". The RNA strands may have the same or different numbers of nucleotides. The maximum number of base pairs in the duplex is the number of nucleotides in the shortest strand of the dsRNA minus any overhangs present in the duplex. In addition to duplex structures, RNAi constructs may also include one or more nucleotide overhangs.

In other embodiments, the sense and antisense strands that hybridize to form the duplex region may be part of a single RNA molecule, i.e., the sense and antisense strands are part of the self-complementary region of a single RNA molecule. In such cases, a single RNA molecule comprises a duplex region (also referred to as a stem region) and a loop region. The 3 'end of the sense strand is joined to the 5' end of the antisense strand by a continuous unpaired nucleotide sequence forming a loop region. The loop region is typically of sufficient length to allow the RNA molecule to fold back upon itself so that the antisense strand can base pair with the sense strand to form a duplex or stem region. The loop region may comprise from about 3 to about 25, from about 5 to about 15, or from about 8 to about 12 unpaired nucleotides. Such RNA molecules having at least partially self-complementary regions are referred to as "short hairpin RNAs" (shrnas). In some embodiments, the loop region may comprise at least 1, 2, 3, 4, 5, 10, 20, or 25 unpaired nucleotides. In some embodiments, the loop region may have 10, 9, 8, 7, 6, 5, 4, 3, 2, or fewer unpaired nucleotides. In certain embodiments, RNAi constructs of the invention comprise shRNA. The individual at least partially self-complementary RNA molecules can be about 35 nucleotides to about 100 nucleotides, about 45 nucleotides to about 85 nucleotides, or about 50 to about 60 nucleotides in length, and comprise a duplex region and a loop region, each region having a length as recited herein.

In some embodiments, RNAi constructs of the invention comprise a sense strand and an antisense strand, wherein the antisense strand comprises a region having a sequence substantially or completely complementary to an HSD17B13 messenger RNA (mRNA) sequence. As used herein, "HSD17B13 mRNA sequence" refers to any messenger RNA sequence, including splice variants, that encode HSD17B13 proteins, including variants or isoforms of HSD17B13 proteins from any species (e.g., mouse, rat, non-human primate, human).

The HSD17B13 mRNA sequence also includes transcribed sequences expressed as its complementary DNA (cDNA) sequence. cDNA sequence refers to a sequence of mRNA transcripts expressed as DNA bases (e.g., guanine, adenine, thymine, and cytosine) rather than RNA bases (e.g., guanine, adenine, uracil, and cytosine). Thus, the antisense strand of an RNAi construct of the invention can comprise a region having a sequence that is substantially or completely complementary to the target HSD17B13 mRNA sequence or HSD17B13cDNA sequence. The HSD17B13 mRNA or cDNA sequence may include, but is not limited to, any HSD17B13 mRNA or cDNA sequence, for example, may be derived from NCBI reference sequences nm_178135.4 or nm_001136230.2.

The region of the antisense strand may be substantially complementary or fully complementary to at least 15 consecutive nucleotides of the HSD17B13 mRNA sequence. In some embodiments, the target region of the HSD17B13 mRNA sequence (the antisense strand of which comprises the complementary region) can range from about 15 to about 30 consecutive nucleotides, from about 16 to about 28 consecutive nucleotides, from about 18 to about 26 consecutive nucleotides, from about 17 to about 24 consecutive nucleotides, from about 19 to about 25 consecutive nucleotides, from about 19 to about 23 consecutive nucleotides, or from about 19 to about 21 consecutive nucleotides. In certain embodiments, the region comprising the antisense strand of a sequence substantially or fully complementary to the HSD17B13 mRNA sequence may in some embodiments comprise at least 15 contiguous nucleotides from an antisense sequence listed in table 1 or table 2. In other embodiments, the antisense sequence comprises at least 16, at least 17, at least 18, or at least 19 consecutive nucleotides from an antisense sequence listed in table 1 or table 2. In some embodiments, the sense and/or antisense sequences comprise at least 15 nucleotides from the sequences listed in table 1 or 2 and having no more than 1, 2, or 3 nucleotide mismatches.

The sense strand of an RNAi construct typically comprises a sequence sufficiently complementary to the sequence of the antisense strand such that the two strands hybridize under physiological conditions to form a duplex region. "duplex region" refers to a region in two complementary or substantially complementary polynucleotides that form base pairs with each other by Watson-Crick base pairing or other hydrogen bonding interactions to create a duplex between the two polynucleotides. The duplex region of the RNAi construct should be of sufficient length to allow the RNAi construct to enter the RNA interference pathway, such as by using Dicer enzyme and/or RISC complex. For example, in some embodiments, the duplex region is about 15 to about 30 base pairs in length. Other lengths of duplex regions within this range are also suitable, such as about 15 to about 28 base pairs, about 15 to about 26 base pairs, about 15 to about 24 base pairs, about 15 to about 22 base pairs, about 17 to about 28 base pairs, about 17 to about 26 base pairs, about 17 to about 24 base pairs, about 17 to about 23 base pairs, about 17 to about 21 base pairs, about 19 to about 25 base pairs, about 19 to about 23 base pairs, or about 19 to about 21 base pairs. In one embodiment, the duplex region is about 17 to about 24 base pairs in length. In another embodiment, the duplex region is about 19 to about 21 base pairs in length.

In some embodiments, RNAi constructs of the invention contain a duplex region of about 24 to about 30 nucleotides that interacts with a target RNA sequence (e.g., HSD17B13 target mRNA sequence) to direct cleavage of the target RNA. Without wishing to be bound by theory, long double stranded RNA introduced into cells can be broken down into siRNA by a type III endonuclease called Dicer (Sharp et al (2001) Genes Dev. [ Gene and development ] 15:485). Dicer (ribonuclease-III-like enzyme) processes dsRNA into 19-23 base pair short interfering RNA with a characteristic two base 3' overhang (Bernstein et al (2001) Nature [ Nature ] 409:363). The siRNA is then incorporated into an RNA-induced silencing complex (RISC), in which one or more helices cleave the siRNA duplex, enabling the complementary antisense strand to direct target recognition (Nykanen et al, (2001) Cell [ Cell ] 107:309). Upon binding to the appropriate target mRNA, one or more endonucleases within RISC cleave the target to induce silencing (Elbashir et al (2001) Genes Dev. [ Gene and development ] 15:188).

For embodiments in which the sense strand and the antisense strand are two separate molecules (e.g., the RNAi construct comprises siRNA), the length of the sense strand and the antisense strand need not be the same as the length of the duplex region. For example, one or both strands may be longer than the duplex region and have one or more unpaired nucleotides or mismatches flanking the duplex region. Thus, in some embodiments, the RNAi construct comprises at least one nucleotide overhang. As used herein, "nucleotide overhang" refers to one or more unpaired nucleotides or nucleotides that extend beyond the strand end of the duplex region. Nucleotide overhangs are typically created when the 3 'end of one strand extends beyond the 5' end of the other strand or when the 5 'end of one strand extends beyond the 3' end of the other strand. The length of the nucleotide overhang is typically between 1 and 6 nucleotides, between 1 and 5 nucleotides, between 1 and 4 nucleotides, between 1 and 3 nucleotides, between 2 and 6 nucleotides, between 2 and 5 nucleotides, or between 2 and 4 nucleotides. In some embodiments, the nucleotide overhang comprises 1, 2, 3, 4, 5, or 6 nucleotides. In a particular embodiment, the nucleotide overhang comprises 1 to 4 nucleotides. In certain embodiments, the nucleotide overhang comprises 2 nucleotides. The nucleotides in the overhangs may be ribonucleotides, deoxyribonucleotides or modified nucleotides as described herein. In some embodiments, the overhang comprises a 5 '-uridine-3' (5 '-UU-3') dinucleotide. In such embodiments, the UU dinucleotide may comprise a ribonucleotide or a modified nucleotide, e.g., a 2' -modified nucleotide. In other embodiments, the overhang comprises a 5 '-deoxythymidine-3' (5 '-dTdT-3') dinucleotide.

Nucleotide overhangs may be located at the 5 'or 3' end of one or both strands. For example, in one embodiment, the RNAi construct comprises nucleotide overhangs at the 5 'and 3' ends of the antisense strand. In another embodiment, the RNAi construct comprises nucleotide overhangs at the 5 'and 3' ends of the sense strand. In some embodiments, the RNAi construct comprises a nucleotide overhang at the 5 'end of the sense strand and the 5' end of the antisense strand. In other embodiments, the RNAi construct comprises a nucleotide overhang at the 3 'end of the sense strand and the 3' end of the antisense strand.

RNAi constructs may comprise a single nucleotide overhang at one end of the double stranded RNA molecule and a blunt end at the other end. By "blunt end" is meant that the sense strand and the antisense strand are fully base paired at the molecular end and no unpaired nucleotide extends beyond the duplex region. In some embodiments, the RNAi construct comprises a nucleotide overhang at the 3' end of the sense strand and a blunt end at the 5' end of the sense strand and the 3' end of the antisense strand. In other embodiments, the RNAi construct comprises a nucleotide overhang at the 3' end of the antisense strand and a blunt end at the 5' end of the antisense strand and the 3' end of the sense strand. In certain embodiments, the RNAi construct comprises blunt ends at both ends of the double-stranded RNA molecule. In such embodiments, the sense strand and the antisense strand have the same length, and the duplex region is the same length as the sense strand and the antisense strand (i.e., the molecule is double-stranded throughout its length).

The sense strand and the antisense strand can each independently have about 15 to about 30 nucleotides in length, about 18 to about 28 nucleotides in length, about 19 to about 27 nucleotides in length, about 19 to about 25 nucleotides in length, about 19 to about 23 nucleotides in length, about 21 to about 25 nucleotides in length, or about 21 to about 23 nucleotides in length. In certain embodiments, the sense strand and the antisense strand are each about 18, about 19, about 20, about 21, about 22, about 23, about 24, or about 25 nucleotides in length. In some embodiments, the sense strand and the antisense strand have the same length, but are formed shorter than those strands such that the RNAi construct has a duplex region with two nucleotide overhangs. For example, in one embodiment, the RNAi construct comprises (i) a sense strand and an antisense strand each 21 nucleotides in length, (ii) a duplex region 19 base pairs in length, and (iii) a nucleotide overhang of 2 unpaired nucleotides at the 3 'end of the sense strand and the 3' end of the antisense strand. In another embodiment, the RNAi construct comprises (i) a sense strand and an antisense strand each 23 nucleotides in length, (ii) a duplex region 21 base pairs in length, and (iii) a nucleotide overhang of 2 unpaired nucleotides at the 3 'end of the sense strand and the 3' end of the antisense strand. In other embodiments, the sense strand and the antisense strand have the same length and form a duplex region over their entire length such that there are no nucleotide overhangs on either end of the duplex molecule. In one such embodiment, the RNAi construct is blunt-ended and comprises (i) a sense strand and an antisense strand each 21 nucleotides in length and (ii) a duplex region 21 base pairs in length. In another such embodiment, the RNAi construct is blunt-ended and comprises (i) a sense strand and an antisense strand each 23 nucleotides in length and (ii) a duplex region 23 base pairs in length.

In other embodiments, the sense strand or antisense strand is longer than the other strand, and both strands form a duplex region that is equal in length to the shorter strand such that the RNAi construct comprises at least one nucleotide overhang. For example, in one embodiment, the RNAi construct comprises (i) a sense strand 19 nucleotides in length, (ii) an antisense strand 21 nucleotides in length, (iii) a duplex region 19 base pairs in length, and (iv) a single nucleotide overhang of 2 unpaired nucleotides at the 3' end of the antisense strand. In another embodiment, the RNAi construct comprises (i) a sense strand 21 nucleotides in length, (ii) an antisense strand 23 nucleotides in length, (iii) a duplex region 21 base pairs in length, and (iv) a single nucleotide overhang of 2 unpaired nucleotides at the 3' end of the antisense strand.

The antisense strand of an RNAi construct of the invention can comprise the sequence of any one of the antisense sequences listed in table 1 or table 2 or the sequence of nucleotides 1-19 of any of these antisense sequences. Each antisense sequence listed in tables 1 and 6 comprises a sequence of 19 consecutive nucleotides (the first 19 nucleotides counted from the 5' end) that is complementary to the HSD17B13 mRNA sequence plus the two nucleotide overhang sequence. Thus, in some embodiments, the antisense strand comprises the sequence of nucleotides 1-19 of any one of SEQ ID NOS: 1-646 or 648-1292.

Modified nucleotides

RNAi constructs of the invention may comprise one or more modified nucleotides. "modified nucleotide" refers to a nucleotide having one or more chemical modifications to a nucleoside, nucleobase, pentose ring, or phosphate group. As used herein, modified nucleotides do not encompass ribonucleotides that contain adenosine monophosphate, guanosine monophosphate, uridine monophosphate, and cytidine monophosphate, as well as deoxyribonucleotides that contain deoxyadenosine monophosphate, deoxyguanosine monophosphate, deoxythymidine monophosphate, and deoxycytidine monophosphate. However, RNAi constructs can comprise a combination of modified nucleotides, ribonucleotides, and deoxyribonucleotides. Incorporation of modified nucleotides into one or both strands of a double-stranded RNA molecule can improve the in vivo stability of the RNA molecule, for example by reducing the susceptibility of the molecule to nucleases and other degradation processes. The effectiveness of RNAi constructs in reducing expression of target genes can also be enhanced by incorporation of modified nucleotides.

In certain embodiments, the modified nucleotide has a modification of ribose. These sugar modifications may include modifications at the 2 'and/or 5' positions of the pentose ring and bicyclic sugar modifications. A2 '-modified nucleotide refers to a nucleotide having a pentose ring with a substituent other than H or OH at the 2' -position. Such 2' modifications include, but are not limited to, 2' -O-alkyl (e.g., O-C1-C10 or O-C1-C10 substituted alkyl), 2' -O-allyl (O-ch2ch=ch2), 2' -C-allyl, 2' -fluoro, 2' -O-methyl (OCH 3), 2' -O-methoxyethyl (O- (CH 2) 2OCH 3), 2' -OCF3, 2' -O (CH 2) 2SCH3, 2' -O-aminoalkyl, 2' -amino (e.g., NH 2), 2' -O-ethylamine, and 2' -azido. Modifications at the 5' position of the pentose ring include, but are not limited to: 5' -methyl (R or S); 5 '-vinyl and 5' -methoxy.

"bicyclic sugar modification" refers to modification of a pentose ring in which a bridging group connects two atoms of the ring to form a second ring, resulting in a bicyclic sugar structure. In some embodiments, the bicyclic sugar modification comprises a bridging group between the 4 'and 2' carbons of the pentose ring. Nucleotides comprising a sugar moiety with a bicyclic sugar modification are referred to herein as bicyclic nucleic acids or BNA. Exemplary bicyclic sugar modifications include, but are not limited to, α -L-methyleneoxy (4 '-CH 2-O-2') Bicyclic Nucleic Acid (BNA); beta-D-methyleneoxy (4 '-CH 2-O-2') BNA (also known as locked nucleic acid or LNA); ethyleneoxy (4 '- (CH 2) 2-O-2') BNA; aminooxy (4 '-CH2-O-N (R) -2') BNA; oxyamino (4 '-CH2-N (R) -O-2') BNA; methyl (methyleneoxy) (4 '-CH (CH 3) -O-2') BNA (also known as constrained ethyl or cEt); methylene-thio (4 '-CH 2-S-2') BNA; methylene-amino (4 '-CH2-N (R) -2') BNA; methyl carbocycle (4 '-CH2-CH (CH 3) -2') BNA; propylene carbocycle (4 '- (CH 2) 3-2') BNA; and methoxy (ethyleneoxy) (4 '-CH (CH 2 OMe) -O-2') BNA (also known as restricted MOE or cMOE). These and other sugar-modified nucleotides that may be incorporated into the RNAi constructs of the invention are described in U.S. Pat. No. 9,181,551, U.S. patent publication No. 2016/012761, and Deleavey and Damha, chemistry and Biology [ chemical and biological ], vol.19:937-954,2012, all of which are incorporated herein by reference in their entirety.

In some embodiments, the RNAi construct comprises one or more 2 '-fluoro-modified nucleotides, 2' -O-methyl-modified nucleotides, 2 '-O-methoxyethyl-modified nucleotides, 2' -O-allyl-modified nucleotides, bicyclic Nucleic Acids (BNA), diol nucleic acids, or a combination thereof. In certain embodiments, the RNAi construct comprises one or more 2' -fluoro-modified nucleotides, 2' -O-methyl-modified nucleotides, 2' -O-methoxyethyl-modified nucleotides, or a combination thereof. In a particular embodiment, the RNAi construct comprises one or more 2 '-fluoro modified nucleotides, 2' -O-methyl modified nucleotides, or a combination thereof.

Both the sense and antisense strands of an RNAi construct can comprise one or more modified nucleotides. For example, in some embodiments, the sense strand comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more modified nucleotides. In certain embodiments, all nucleotides in the sense strand are modified nucleotides. In some embodiments, the antisense strand comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more modified nucleotides. In other embodiments, all nucleotides in the antisense strand are modified nucleotides. In certain other embodiments, all nucleotides in the sense strand and all nucleotides in the antisense strand are modified nucleotides. In these and other embodiments, the modified nucleotide may be a 2 '-fluoro modified nucleotide, a 2' -O-methyl modified nucleotide, or a combination thereof.

In some embodiments, all pyrimidine nucleotides preceding the adenosine nucleotide in the sense, antisense, or both strands are modified nucleotides. For example, when the sequence 5' -CA-3' or 5' -UA-3' is present in either strand, cytidine and uridine nucleotides are modified nucleotides, preferably nucleotides modified with a 2' -O-methyl group. In certain embodiments, all pyrimidine nucleotides in the sense strand are modified nucleotides (e.g., 2' -O-methyl modified nucleotides), and the 5' nucleotide in all occurrences of the sequence 5' -CA-3' or 5' -UA-3' in the antisense strand is a modified nucleotide (e.g., a 2' -O-methyl modified nucleotide). In other embodiments, all nucleotides in the duplex region are modified nucleotides. In such embodiments, the modified nucleotide is preferably a 2 '-O-methyl modified nucleotide, a 2' -fluoro modified nucleotide, or a combination thereof.

In embodiments where the RNAi construct comprises a nucleotide overhang, the nucleotide in the overhang can be a ribonucleotide, a deoxyribonucleotide, or a modified nucleotide. In one embodiment, the nucleotides in the overhangs are deoxyribonucleotides, such as deoxythymidine. In another embodiment, the nucleotides in the overhang are modified nucleotides. For example, in some embodiments, the nucleotides in the overhang are 2' -O-methyl modified nucleotides, 2' -fluoro modified nucleotides, 2' -methoxyethyl modified nucleotides, or a combination thereof.

RNAi constructs of the invention may also comprise one or more modified internucleotide linkages. As used herein, the term "modified internucleotide linkage" refers to internucleotide linkages other than the natural 3 'to 5' phosphodiester linkages. In some embodiments, the modified internucleotide linkages are phosphorus-containing internucleotide linkages, such as phosphotriesters, aminoalkyl phosphotriesters, alkyl phosphonates (e.g., methyl phosphonate, 3 '-alkylene phosphonate), phosphinates, phosphoramidates (e.g., 3' -phosphoramidate and aminoalkyl phosphoramidate), phosphorothioates (p=s), chiral phosphorothioates, phosphorodithioates, phosphorothioates, alkyl phosphorothioates, and borane phosphates. In one embodiment, the modified internucleotide linkage is a 2 'to 5' phosphodiester linkage. In other embodiments, the modified internucleotide linkages are phosphorus-free internucleotide linkages, and thus may be referred to as modified internucleoside linkages. Such non-phosphorus containing linkages include, but are not limited to, morpholine linkages (formed in part from the sugar portion of the nucleoside); siloxane bond (-O-Si (H) 2-O-); sulfide, sulfoxide, and sulfone linkages; formyl and thiocarbonyl linkages; an alkene-containing backbone; sulfamate backbone; methylene methylimino (-CH 2-N (CH 3) -O-CH 2-) and methylene hydrazino linkages; sulfonate and sulfonamide linkages; an amide bond; and other bonds with mixed N, O, S and CH2 component moieties. In one embodiment, the modified internucleoside linkages are peptide-based linkages (e.g., aminoethylglycine) that result in peptide nucleic acids or PNAs, such as those described in U.S. Pat. nos. 5,539,082, 5,714,331, and 5,719,262. Other suitable modified internucleotide and internucleoside linkages that can be used in RNAi constructs of the invention are described in U.S. Pat. No. 6,693,187, U.S. Pat. No. 9,181,551, U.S. patent publication No. 2016/012761 and Deleavey and Damha, chemistry and Biology [ chemistry and biology ], volume 19:937-954,2012, all of which are hereby incorporated by reference in their entirety.

In certain embodiments, the RNAi construct comprises one or more phosphorothioate internucleotide linkages. Phosphorothioate internucleotide linkages may be present in the sense, antisense, or both strands of an RNAi construct. For example, in some embodiments, the sense strand comprises 1, 2, 3, 4, 5, 6, 7, 8, or more phosphorothioate internucleotide linkages. In other embodiments, the antisense strand comprises 1, 2, 3, 4, 5, 6, 7, 8 or more phosphorothioate internucleotide linkages. In still other embodiments, both strands comprise 1, 2, 3, 4, 5, 6, 7, 8 or more phosphorothioate internucleotide linkages. RNAi constructs may comprise one or more phosphorothioate internucleotide linkages at the 3 '-terminus, 5' -terminus, or both the 3 '-and 5' -termini of the sense, antisense, or both strands. For example, in certain embodiments, the RNAi construct comprises about 1 to about 6 or more (e.g., about 1, 2, 3, 4, 5, 6 or more) continuous phosphorothioate internucleotide linkages at the 3' -end of the sense strand, antisense strand, or both strands. In other embodiments, the RNAi construct comprises about 1 to about 6 or more (e.g., about 1, 2, 3, 4, 5, 6 or more) continuous phosphorothioate internucleotide linkages at the 5' -end of the sense strand, antisense strand, or both strands. In one embodiment, the RNAi construct comprises a single phosphorothioate internucleotide linkage at the 3 'end of the sense strand and a single phosphorothioate internucleotide linkage at the 3' end of the antisense strand. In another embodiment, the RNAi construct comprises two consecutive phosphorothioate internucleotide linkages at the 3 'end of the antisense strand (i.e., phosphorothioate internucleotide linkages at the first and second internucleotide linkages at the 3' end of the antisense strand). In another embodiment, the RNAi construct comprises two consecutive phosphorothioate internucleotide linkages at the 3 'and 5' ends of the antisense strand. In yet another embodiment, the RNAi construct comprises two consecutive phosphorothioate internucleotide linkages at the 3' and 5' ends of the antisense strand and two consecutive phosphorothioate internucleotide linkages at the 5' end of the sense strand. In another embodiment, the RNAi construct comprises two consecutive phosphorothioate internucleotide linkages at the 3 'and 5' ends of the antisense strand and two consecutive phosphorothioate internucleotide linkages at the 3 'and 5' ends of the sense strand (i.e., phosphorothioate internucleotide linkages at the first and second internucleotide linkages at the 5 'and 3' ends of the antisense strand and phosphorothioate internucleotide linkages at the first and second internucleotide linkages at the 5 'and 3' ends of the sense strand). In any embodiment where one or both strands comprise one or more phosphorothioate internucleotide linkages, the remaining internucleotide linkages within the strand may be natural 3 'to 5' phosphodiester linkages. For example, in some embodiments, each internucleotide linkage of the sense strand and the antisense strand is selected from the group consisting of phosphodiester and phosphorothioate, wherein at least one internucleotide linkage is phosphorothioate.

In embodiments where the RNAi construct comprises nucleotide overhangs, two or more unpaired nucleotides in the overhangs can be linked by phosphorothioate internucleotide linkages. In certain embodiments, all unpaired nucleotides in the nucleotide overhangs at the 3' end of the antisense strand and/or sense strand are linked by phosphorothioate internucleotide linkages. In other embodiments, all unpaired nucleotides in the nucleotide overhangs at the 5' end of the antisense strand and/or sense strand are linked by phosphorothioate internucleotide linkages. In still other embodiments, all unpaired nucleotides in any nucleotide overhang are linked by phosphorothioate internucleotide linkages.

In certain embodiments, the modified nucleotide incorporated into one or both strands of the RNAi constructs of the invention has a modification of a nucleobase (also referred to herein as a "base"). "modified nucleobase" or "modified base" refers to bases other than the naturally occurring purine bases adenine (A) and guanine (G) and the pyrimidine bases thymine (T), cytosine (C) and uracil (U). The modified nucleobases may be synthetic or naturally occurring modifications, including but not limited to: universal bases (universal base), 5-methylcytosine (5-me-C), 5-hydroxymethylcytosine, xanthine (X), hypoxanthine (I), 2-aminoadenine, 6-methyladenine, 6-methylguanine, other alkyl derivatives of adenine and guanine, 2-propyl and other alkyl derivatives of adenine and guanine, 2-thiouracil, 2-thiothymine and 2-thiocytosine, 5-halouracil and cytosine, 5-propynyluracil and cytosine, 6-azouracil, cytosine and thymine, 5-uracil (pseudouracil), 4-thiouracil, 8-halo, 8-amino, 8-mercapto, 8-thioalkyl, 8-hydroxy and other 8-substituted adenine and guanine, 5-halo, in particular 5-bromo, 5-trifluoromethyl and other 5-substituted uracil and cytosine, 7-methyladenine and 7-methyladenine, 8-azaguanine and 8-azaguanine, 7-deazaadenine and 7-deazaadenine (3 'and 3-deazaadenine are absent or absent base and 3' deazanucleotide residues are absent, and may be an inverted nucleotide of any of the above, including inverted abasic nucleotides and inverted deoxynucleotides).

In some embodiments, the modified base is a universal base. "universal base" refers to a base analog that forms base pairs indiscriminately from all natural bases in RNA and DNA without altering the duplex structure of the resulting duplex region. Universal bases are known to those of ordinary skill in the art and include, but are not limited to, inosine, C-phenyl, C-naphthyl, and other aromatic derivatives, azole carboxamides, and nitroazole derivatives, such as 3-nitropyrrole, 4-nitroindole, 5-nitroindole, and 6-nitroindole.

Other suitable modified bases that may be incorporated into the RNAi constructs of the invention include those described in Herdewijn, antisense Nucleic Acid Drug Dev [ antisense nucleic acid drug development ], vol.10:297-310, 2000 and Peacok et al, J.org.Chern [ journal of organic chemistry ], vol.76:7295-7300, 2011, which are hereby incorporated by reference in their entirety. It is well understood by those of ordinary skill in the art that guanine, cytosine, adenine, thymine, and uracil can be replaced with other nucleobases, such as the modified nucleobases described above, without substantially altering the base pairing properties of a polynucleotide comprising a nucleotide carrying the substituted nucleobase.

In some embodiments of the RNAi constructs of the invention, the sense, antisense, or 5' ends of the antisense and sense strands comprise a phosphate moiety. As used herein, the term "phosphate moiety" is meant to include unmodified phosphate (-O-p=o) (OH) as well as terminal phosphate groups of modified phosphate. Modified phosphates include phosphates wherein one or more O and OH groups are substituted with H, O, S, N (R) or alkyl, wherein R is H, an amino protecting group or unsubstituted or substituted alkyl. Exemplary phosphate moieties include, but are not limited to: 5' -monophosphate; 5' -diphosphate; 5' -triphosphate; a 5' -guanosine cap (7-methylated or unmethylated); a 5' -adenosine cap or any other modified or unmodified nucleotide cap structure; 5' -monothiophosphate (phosphorothioate); 5' -mono-dithiophosphate (dithiophosphate); 5' - α -thiotriphosphate; 5 '-gamma-thiophosphoric acid ester, 5' -phosphoramidate; 5' -vinyl phosphonate; 5' -alkylphosphonates (e.g., alkyl = methyl, ethyl, isopropyl, propyl, etc.); and 5' -alkyl ether phosphonates (e.g., alkyl ether = methoxymethyl, ethoxymethyl, etc.).

Modified nucleotides that may be incorporated into RNAi constructs of the invention can have more than one chemical modification described herein. For example, the modified nucleotide may have a modification to ribose and a modification to nucleobase. For example, a modified nucleotide may comprise a 2' sugar modification (e.g., 2' -fluoro or 2' -methyl) and comprise a modified base (e.g., 5-methylcytosine or pseudouracil). In other embodiments, the modified nucleotides may comprise sugar modifications as well as modifications to the 5' phosphate that, when incorporated into a polynucleotide, will result in modified internucleotide or internucleoside linkages. For example, in some embodiments, the modified nucleotide may comprise a sugar modification, such as a 2' -fluoro modification, a 2' -O-methyl modification, or a bicyclic sugar modification, as well as a 5' phosphorothioate group. Thus, in some embodiments, one or both strands of an RNAi construct of the invention comprises a 2' modified nucleotide or a combination of BNA and phosphorothioate internucleotide linkages. In certain embodiments, both the sense and antisense strands of the RNAi constructs of the invention comprise a combination of 2 '-fluoro modified nucleotides, 2' -O-methyl modified nucleotides, and phosphorothioate internucleotide linkages. Exemplary RNAi constructs comprising modified nucleotides and internucleotide linkages are shown in table 2.

RNAi construct function

Preferably, the RNAi constructs of the invention reduce or inhibit expression of HSD17B13 in cells, particularly liver cells. Thus, in one embodiment, the invention provides a method of reducing HSD17B13 expression in a cell by contacting the cell with any of the RNAi constructs described herein. The cells may be in vitro or in vivo. HSD17B13 expression can be assessed by measuring the amount or level of HSD17B13 mRNA, HSD17B13 protein, or another biomarker associated with HSD17B13 expression. The reduction in HSD17B13 expression in cells or animals treated with the RNAi constructs of the invention can be determined relative to HSD17B13 expression in cells or animals not treated with the RNAi constructs or treated with a control RNAi construct. For example, in some embodiments, the decrease in HSD17B13 expression is assessed by: (a) measuring the amount or level of HSD17B13 mRNA in liver cells treated with an RNAi construct of the invention, (B) measuring the amount or level of HSD17B13 mRNA in liver cells treated with a control RNAi construct (e.g., an RNAi construct directed to an RNA molecule not expressed in liver cells or an RNAi construct having a nonsense or disordered sequence) or not treated with the construct, and (c) comparing the measured HSD17B13 mRNA level from the treated cells in (a) to the measured HSD17B13 mRNA level from the control cells in (B). Prior to comparison, HSD17B13 mRNA levels in the treated cells and control cells can be normalized to the RNA level of the control gene (e.g., 18S ribosomal RNA). HSD17B13 mRNA levels can be measured by a variety of methods including northern blot analysis, nuclease protection assays, fluorescence In Situ Hybridization (FISH), reverse Transcriptase (RT) -PCR, real-time RT-PCR, quantitative PCR, and the like.

In other embodiments, the reduction in HSD17B13 expression is assessed by: (a) measuring the amount or level of HSD17B13 protein in liver cells treated with an RNAi construct of the invention, (B) measuring the amount or level of HSD17B13 protein in liver cells treated with a control RNAi construct (e.g., an RNAi construct directed to an RNA molecule not expressed in liver cells or an RNAi construct having a nonsense or disordered sequence) or not treated with the construct, and (c) comparing the measured HSD17B13 protein level from the treated cells in (a) to the measured HSD17B13 protein level from the control cells in (B). Methods for measuring HSD17B13 protein levels are known to those of ordinary skill in the art and include immunoblotting, immunoassays (e.g., ELISA) and flow cytometry. An exemplary microdroplet digital PCR method for assessing HSD17B13 expression is described in example 2. Any method capable of measuring HSD17B13 mRNA or protein may be used to assess the efficacy of the RNAi constructs of the invention.

In some embodiments, the method of assessing HSD17B13 expression levels is performed in vitro in cells that naturally express HSD17B13 (e.g., liver cells) or cells that have been engineered to express HSD17B 13. In certain embodiments, these methods are performed in vitro in liver cells. Suitable liver cells include, but are not limited to: primary hepatocytes (e.g., human, non-human primate, or rodent hepatocytes), hepAD38 cells, huH-6 cells, huH-7 cells, huH-5-2 cells, BNLCL2 cells, hep3B cells, or HepG2 cells.

In other embodiments, the method of assessing HSD17B13 expression levels is performed in vivo. The RNAi construct and any control RNAi construct can be administered to an animal (e.g., rodent or non-human primate) and after treatment, HSD17B13 mRNA or protein levels are assessed in liver tissue harvested from the animal. Alternatively or additionally, biomarkers or functional phenotypes associated with HSD17B13 expression may be assessed in treated animals.

In certain embodiments, expression of HSD17B13 is reduced by at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, or at least 50% in liver cells by an RNAi construct of the invention. In some embodiments, expression of HSD17B13 is reduced by at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, or at least 85% in liver cells by an RNAi construct of the invention. In other embodiments, expression of HSD17B13 is reduced by about 90% or more, e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more, in liver cells by an RNAi construct of the invention. The percent reduction in HSD17B13 expression can be measured by any of the methods described herein, as well as other methods known in the art. For example, in certain embodiments, RNAi constructs of the invention inhibit HSD17B13 expression by at least 70% in vitro in primary hepatocytes (expressing wild-type HSD17B 13) at 5 nM. In related embodiments, RNAi constructs of the invention inhibit HSD17B13 expression by at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, or at least 75% in vitro at 5 nM. In other embodiments, RNAi constructs of the invention inhibit HSD17B13 expression in primary hepatocytes by at least 80%, at least 85%, at least 90%, at least 92%, at least 94%, at least 96%, or at least 98% in vitro at 5 nM. As described in example 2, the decrease in HSD17B13 can be measured using a variety of techniques, including RNA FISH or microdroplet digital PCR.

In some embodiments, IC50 values are calculated to assess the efficacy of RNAi constructs of the invention in inhibiting HSD17B13 expression in liver cells. "IC50 value" is the dose/concentration required to achieve 50% inhibition of biological or biochemical function or level. The IC50 value for any particular substance or antagonist can be determined by constructing a dose-response curve and examining the effect of different concentrations of the substance or antagonist on expression levels or functional activity in any assay. The IC50 value for a given antagonist or substance can be calculated by determining the concentration required to inhibit half of the maximum biological response or natural expression level. Thus, the IC50 value of any RNAi construct can be calculated by determining the concentration of RNAi construct required to inhibit half of the natural HSD17B13 expression level in a liver cell (e.g., control HSD17B13 expression level in a liver cell) in any assay, such as an immunoassay or RNA FISH assay or microdroplet digital PCR assay described in the examples. The RNAi constructs of the invention can inhibit HSD17B13 expression in liver cells (e.g., primary hepatocytes) with an IC50 of less than about 40 nM. For example, the RNAi construct inhibits HSD17B13 expression in liver cells with the following IC 50: about 0.001nM to about 40nM, about 0.001nM to about 30nM, about 0.001nM to about 20nM, about 0.001nM to about 15nM, about 0.1nM to about 10nM, about 0.1nM to about 5nM, or about 0.1nM to about 1nM.

RNAi constructs of the invention can be readily made using techniques known in the art, for example, using conventional nucleic acid solid phase synthesis. Polynucleotides of RNAi constructs can be assembled on a suitable nucleic acid synthesizer using standard nucleotides or nucleoside precursors (e.g., phosphoramidites). Automated nucleic acid synthesizers are commercially available from several suppliers, including DNA/RNA synthesizers from applied biosystems (Applied Biosystems) (foster city, california), merMade synthesizers from bioautomations (bioautomations) (euclidean, texas), and OligoPilot synthesizers from the general electric medical community department of life (GE Healthcare Life Sciences) (pittsburgh, pennsylvania).

The 2 'silyl protecting group can be used in combination with Dimethoxytrityl (DMT) at the 5' acid labile of ribonucleoside to synthesize oligonucleotides by phosphoramidite chemistry. The final deprotection conditions are known not to significantly degrade the RNA products. All syntheses can be carried out on large, medium and small scales in any automated or manual synthesizer. Synthesis may also be performed in multiple well plates, columns or slides.

The 2' -O-silyl group may be removed via exposure to fluoride ions, which may include any fluoride ion source, such as those salts containing fluoride ions paired with inorganic counter ions, such as cesium fluoride and potassium fluoride, or those salts containing fluoride ions paired with organic counter ions, such as tetraalkylammonium fluoride. Crown ether catalysts can be used in combination with inorganic fluorides in the deprotection reaction. Preferred fluoride ion sources are tetrabutylammonium fluoride or aminohydrofluoride (e.g., aqueous HF in combination with triethylamine in a dipolar aprotic solvent such as dimethylformamide).

The choice of protecting groups for the phosphite triesters and the triesters can alter the stability of the triesters to fluoride. Methyl protection of the phosphotriester or phosphite triester may stabilize the linkage with fluoride ions and improve process yields.

Since ribonucleosides have a reactive 2' hydroxyl substituent, it may be desirable to protect the reactive 2' position in the RNA with a protecting group orthogonal to the 5' -O-dimethoxytrityl protecting group (e.g., a protecting group that is stable to treatment with an acid). Silyl protecting groups meet this criteria and can be easily removed during the final fluoride deprotection step, which can result in minimal RNA degradation.

Tetrazole catalysts can be used for standard phosphoramidite coupling reactions. Preferred catalysts include, for example, tetrazole, S-ethyl-tetrazole, benzylthiotetrazole, p-nitrophenyltetrazole.

As will be appreciated by those of ordinary skill in the art, other methods of synthesizing the RNAi constructs described herein will be apparent to those of ordinary skill in the art. Alternatively, the various synthetic steps may be performed in alternating sequence or order to obtain the desired compound. Other synthetic chemical transformations, protecting groups (e.g., for hydroxyl groups, amino groups, etc. present on the base), and protecting group methods (protection and deprotection) that can be used to synthesize the RNAi constructs described herein are known in the art and include, for example, those described in the following: larock, comprehensive Organic Transformations [ full organic transformation ], VCH Publishers [ VCH Publishers ] (1989); T.W.Greene and P.G.M.Wuts, protective Groups in Organic Synthesis [ protecting group in organic Synthesis ], 2 nd edition, john Wiley and Sons [ John Weili father-son company ], (1991); fieser and M.Fieser, fieser and Fieser's Reagents for Organic Synthesis [ Fei Saier and Fei Saier reagents for organic synthesis ], john Wiley and Sons [ John Weili father-son company ] (1994); and L.Paquette edit Encyclopedia of Reagents for Organic Synthesis [ encyclopedia of organic synthetic reagents ], john Wiley and Sons [ John Weili father-son company ] (1995), and subsequent versions thereof. Custom synthesis of RNAi constructs is also available from several commercial suppliers including Dharmacon (Dharmacon, inc.) (lafeet, corrado), AXO labs GmbH (columbach, germany), and Ambion (Ambion, inc.) (foster city, california).

RNAi constructs of the invention may comprise a ligand. As used herein, "ligand" refers to any compound or molecule capable of interacting directly or indirectly with another compound or molecule. Interaction of a ligand with another compound or molecule may trigger a biological response (e.g., initiate a signaling cascade, induce receptor-mediated endocytosis) or may simply be physical association. The ligand may modify one or more properties of the attached double stranded RNA molecule, such as pharmacodynamics, pharmacokinetics, binding, uptake, cell distribution, cell uptake, charge and/or clearance properties of the RNA molecule.

The ligand may comprise a serum protein (e.g., human serum albumin, low density lipoprotein, globulin), cholesterol moiety, vitamin (biotin, vitamin E, vitamin B12), folic acid moiety, steroid, bile acid (e.g., cholic acid), fatty acid (e.g., palmitic acid, myristic acid), carbohydrate (e.g., dextran, pullulan, chitin, chitosan, inulin, cyclodextrin, or hyaluronic acid), glycoside, phospholipid, or antibody or binding fragment thereof (e.g., an antibody or binding fragment that targets the RNAi construct to a particular cell type, e.g., liver). Other examples of ligands include dyes, intercalators (e.g., acridine), cross-linking agents (e.g., psoralen, mitomycin C), porphyrins (TPPC 4, texaphyrin, sapphyrin), polycyclic aromatic hydrocarbons (e.g., phenazine, dihydrophenazine), artificial endonucleases (e.g., EDTA), lipophilic molecules such as adamantaneacetic acid, 1-pyrenebutyric acid, dihydrotestosterone, 1, 3-di (hexadecyl) glycerol, geranoxyhexyl, hexadecyl glycerol, borneol, menthol, 1, 3-propanediol, heptadecyl, 03- (oleoyl) lithocholic acid, 03- (oleoyl) cholic acid, dimethoxytrityl, or phenoxazine), peptides (e.g., antennapedia, tat peptides, RGD peptides), alkylating agents, polymers such as polyethylene glycol (PEG) (e.g., PEG-40K), polyamino acids, and polyamines (such as spermine, spermidine).

In certain embodiments, the ligand has endosomolytic properties. Endosomolytic ligands facilitate endosomolytic and/or transport of the RNAi constructs or components of the invention from the endosome of the cell to the cytoplasm. The endosomolytic ligand may be a polycationic peptide or peptidomimetic that exhibits pH dependent membrane activity and fusion. In one embodiment, the endosomolytic ligand assumes its active conformation at endosomoph. An "active" conformation is one in which endosomolytic ligands promote endosomolytic and/or the RNAi constructs of the invention, or components thereof, are transported from the endosome of the cell to the cytoplasm. Exemplary endosomolytic ligands include GALA peptides (Subbaao et al, biochemistry [ Biochemistry ], volume 26: 2964-2972, 1987), EALA peptides (Vogel et al, J.Am.Chen.Soc. [ American society of chemistry ], volume 118: 1581-1586, 1996), and derivatives thereof (Turk et al, biochem. Biophys. Acta [ journal of Biochemistry and biophysics ], volume 1559: 56-68,2002). In one embodiment, the endosomolytic component may contain chemical groups (e.g., amino acids) that will undergo a change in charge or protonation in response to a change in pH. The endosomolytic component may be linear or branched.

In some embodiments, the ligand comprises a lipid or other hydrophobic molecule. In one embodiment, the ligand comprises a cholesterol moiety or other steroid. Cholesterol conjugated oligonucleotides have been reported to be more active than their unconjugated counterparts (Manoharan, antisense Nucleic Acid Drug Development [ antisense nucleic acid drug development ], vol.12:103-228, 2002). Ligands comprising cholesterol moieties and other lipids to conjugate with nucleic acid molecules are described in U.S. patent nos. 7,851,615, 7,745,608 and 7,833,992, which are hereby incorporated by reference in their entirety. In another embodiment, the ligand comprises a folate moiety. Polynucleotides conjugated to folic acid moieties can be taken up by cells via receptor-mediated endocytosis pathways. Such folate-polynucleotide conjugates are described in U.S. patent No. 8,188,247, which is hereby incorporated by reference in its entirety.

Given that HSD17B13 is expressed in liver cells (e.g., hepatocytes), in certain embodiments, it is desirable to specifically deliver RNAi constructs to those liver cells. In some embodiments, the RNAi construct can specifically target the liver by using a ligand that binds to or interacts with a protein expressed on the surface of liver cells. For example, in certain embodiments, the ligand may comprise an antigen binding protein (e.g., an antibody or binding fragment thereof (e.g., fab, scFv)) that specifically binds to a receptor expressed on hepatocytes (e.g., like ASGR 1).

In certain embodiments, the ligand comprises a carbohydrate. "carbohydrate" refers to a compound composed of one or more monosaccharide units having at least 6 carbon atoms (which may be linear, branched, or cyclic) and oxygen, nitrogen, or sulfur atoms bonded to each carbon atom. Carbohydrates include, but are not limited to, sugars (e.g., monosaccharides, disaccharides, trisaccharides, tetrasaccharides, and oligosaccharides containing about 4, 5, 6, 7, 8, or 9 monosaccharide units) and polysaccharides such as starch, glycogen, cellulose, and polysaccharide gums. In some embodiments, the carbohydrates incorporated into the ligand are monosaccharides selected from pentoses, hexoses, or heptoses and disaccharides and trisaccharides comprising such monosaccharide units. In other embodiments, the carbohydrate incorporated into the ligand is an amino sugar, such as galactosamine, glucosamine, N-acetyl-galactosamine, and N-acetylglucosamine.

In some embodiments, the ligand comprises a hexose or hexosamine. Hexoses may be selected from glucose, galactose, mannose, fucose or fructose. The hexosamine may be selected from fructosamine, galactosamine, glucosamine or mannosamine. In certain embodiments, the ligand comprises glucose, galactose, galactosamine, or glucosamine. In one embodiment, the ligand comprises glucose, glucosamine or N-acetylglucosamine. In another embodiment, the ligand comprises galactose, galactosamine, or N-acetyl-galactosamine. In a particular embodiment, the ligand comprises N-acetyl-galactosamine. Ligands comprising glucose, galactose and N-acetyl-galactosamine (GalNAc) are particularly effective in targeting compounds to liver cells. See, e.g., D' Souza and Devarajan, J.control Release [ J.control Release ], vol.203:126-139, 2015. Examples of GalNAc-or galactose-containing ligands that can be incorporated into RNAi constructs of the invention are described in U.S. patent No. 7,491,805;8,106,022; and 8,877,917; U.S. patent publication No. 20030130186; and WIPO publication No. WO 2013166155, all of which are hereby incorporated by reference in their entirety.

In certain embodiments, the ligand comprises a multivalent carbohydrate moiety. As used herein, "multivalent carbohydrate moiety" refers to a moiety comprising two or more carbohydrate units capable of independently binding or interacting with other molecules. For example, a multivalent carbohydrate moiety comprises two or more binding domains consisting of carbohydrates, which can bind to two or more different molecules or two or more different sites on the same molecule. The valency of a carbohydrate moiety represents the number of individual binding domains within the carbohydrate moiety. For example, the terms "monovalent", "divalent", "trivalent" and "tetravalent" with respect to a carbohydrate moiety refer to carbohydrate moieties having one, two, three and four binding domains, respectively. The multivalent carbohydrate moiety may comprise a multivalent lactose moiety, a multivalent galactose moiety, a multivalent glucose moiety, a multivalent N-acetyl-galactosamine moiety, a multivalent N-acetyl-glucosamine moiety, a multivalent mannose moiety, or a multivalent fucose moiety. In some embodiments, the ligand comprises a multivalent galactose moiety. In other embodiments, the ligand comprises a multivalent N-acetyl-galactosamine moiety. In these and other embodiments, the multivalent carbohydrate moiety is divalent, trivalent, or tetravalent. In such embodiments, the multivalent carbohydrate moiety may be bi-antennary or tri-antennary. In a particular embodiment, the multivalent N-acetyl-galactosamine moiety is trivalent or tetravalent. In another particular embodiment, the multivalent galactose moiety is trivalent or tetravalent. Exemplary ligands containing trivalent and tetravalent GalNAc for incorporation into RNAi constructs of the invention are described in detail below.

The ligand may be directly or indirectly attached or conjugated to the RNAi construct to the RNA molecule. For example, in some embodiments, the ligand is directly covalently attached to the sense or antisense strand of the RNAi construct. In other embodiments, the ligand is covalently attached to the sense or antisense strand of the RNAi construct via a linker. The ligand may be attached to a nucleobase, sugar moiety, or internucleotide linkage of a polynucleotide (e.g., sense strand or antisense strand) of an RNAi construct of the invention. Conjugation or attachment to the purine nucleobase or derivative thereof may occur at any position including in-and out-of-loop atoms. In certain embodiments, the 2-, 6-, 7-or 8-position of the purine nucleobase is attached to a ligand. Conjugation or attachment to the pyrimidine nucleobase or derivative thereof may also occur at any position. In some embodiments, the pyrimidine nucleobases at the 2-, 5-, and 6-positions may be attached to a ligand. Conjugation or attachment to the sugar moiety of a nucleotide may occur at any carbon atom. Exemplary carbon atoms of the sugar moiety that may be attached to the ligand include 2', 3', and 5' carbon atoms. The 1' position may also be linked to a ligand, for example in a basic residue. Internucleotide linkages may also support ligand attachment. For phosphorus-containing linkages (e.g., phosphodiester, phosphorothioate, phosphorodithioate, phosphoramidate, etc.), the ligand may be attached directly to the phosphorus atom or to a O, N or S atom bonded to the phosphorus atom. For amine-or amide-containing internucleoside linkages (e.g., PNAs), the ligand may be attached to the nitrogen atom of the amine or amide or to an adjacent carbon atom.

In certain embodiments, the ligand may be attached to the 3 'or 5' end of the sense or antisense strand. In certain embodiments, the ligand is covalently linked to the 5' end of the sense strand. In other embodiments, the ligand is covalently linked to the 3' end of the sense strand. For example, in some embodiments, the ligand is attached to the 3' terminal nucleotide of the sense strand. In some such embodiments, the ligand is attached at the 3 'position of the 3' terminal nucleotide of the sense strand. In alternative embodiments, the ligand is attached near the 3' end of the sense strand, but before one or more terminal nucleotides (i.e., before 1, 2, 3, or 4 terminal nucleotides). In some embodiments, the ligand is attached at the 2 'position of the sugar of the 3' terminal nucleotide of the sense strand.

In certain embodiments, the ligand is attached to the sense strand or the antisense strand via a linker. A "linker" is an atom or group of atoms that covalently links the ligand to the polynucleotide component of the RNAi construct. The linker may be about 1 to about 30 atoms in length, about 2 to about 28 atoms in length, about 3 to about 26 atoms in length, about 4 to about 24 atoms in length, about 6 to about 20 atoms in length, about 7 to about 20 atoms in length, about 8 to about 18 atoms in length, about 10 to about 18 atoms in length, and about 12 to about 18 atoms in length. In some embodiments, the linker may comprise a difunctional linking moiety, which typically comprises an alkyl moiety having two functional groups. One functional group is selected to bind to a compound of interest (e.g., the sense strand or antisense strand of an RNAi construct), and the other functional group is selected to bind to substantially any selected group, e.g., a ligand as described herein. In certain embodiments, the linker comprises a chain structure or oligomer of repeating units, such as ethylene glycol or amino acid units. Examples of functional groups typically used for the difunctional linking moiety include, but are not limited to, electrophiles for reacting with nucleophilic groups and nucleophiles for reacting with electrophiles. In some embodiments, the difunctional linking moiety includes amino groups, hydroxyl groups, carboxylic acids, thiols, unsaturated bonds (e.g., double or triple bonds), and the like.

Linkers useful for linking the ligand to the sense or antisense strand in the RNAi constructs of the invention include, but are not limited to, pyrrolidine, 8-amino-3, 6-dioxaoctanoic acid, 4- (N-maleimidomethyl) cyclohexane-1-carboxylic acid succinimidyl ester, 6-aminocaproic acid, substituted C1-C10 alkyl, substituted or unsubstituted C2-C10 alkenyl, or substituted or unsubstituted C2-C10 alkynyl. Preferred substituents for such linkers include, but are not limited to, hydroxy, amino, alkoxy, carboxy, benzyl, phenyl, nitro, thiol, thioalkoxy, halogen, alkyl, aryl, alkenyl, and alkynyl.

In certain embodiments, the linker is cleavable. Cleavable linkers are linkers that are sufficiently stable extracellular, but that are cleaved upon entry into a target cell to release the two parts of the linker that remain together. In some embodiments, the cleavable linker is cleaved at least 10-fold, 20-fold, 30-fold, 40-fold, 50-fold, 60-fold, 70-fold, 80-fold, 90-fold or more, or at least 100-fold faster in the target cell or in a first reference condition (which may, for example, be selected to mimic or represent an intracellular condition) than in the subject's blood or in a second reference condition (which may, for example, be selected to mimic or represent a condition found in blood or serum).

Cleavable linkers are susceptible to cleavage agents such as pH, redox potential, or the presence of degradation molecules. In general, lysing agents are found more commonly or at higher levels or activities within cells than in serum or blood. Examples of such degradation agents include: redox agents selected for a particular substrate or not having substrate specificity, including, for example, an oxidation or reduction enzyme or reducing agent present in the cell, such as a thiol, which can degrade the redox cleavable linker by reduction; an esterase; endosomes or agents that can form acidic environments, such as those that produce a pH of 5 or less; enzymes, peptidases (which may be substrate specific), and phosphatases that hydrolyze or degrade acid cleavable linkers by acting as a general acid.

The cleavable linker may comprise a pH sensitive moiety. The pH of human serum is 7.4, while the average intracellular pH is slightly lower, ranging from about 7.1-7.3. Endosomes have a higher acidic pH in the range of 5.5-6.0, and lysosomes have an even higher acidic pH of about 5.0. Some linkers will have cleavable groups that cleave at a preferred pH, thereby releasing the RNA molecule from the ligand into the cell, or into a desired compartment of the cell.

The linker may comprise a cleavable group cleavable by a specific enzyme. The type of cleavable group incorporated into the linker may depend on the cell to be targeted. For example, the liver targeting ligand may be linked to the RNA molecule through a linker comprising an ester group. Liver cells are rich in esterases and therefore the linker will lyse more efficiently in liver cells than in cell types that are not esterase-rich. Other types of cells rich in esterases include cells of the lung, renal cortex and testes. When targeting peptidase-rich cells (e.g., liver cells and synovial cells), linkers containing peptide bonds may be used.

In general, the suitability of a candidate cleavable linker can be assessed by testing the ability of the degrading agent (or condition) to cleave the candidate linker. It is also desirable to test candidate cleavable linkers for their ability to resist cleavage in blood or when in contact with other non-target tissues. Thus, a relative susceptibility to lysis may be determined between a first condition selected to indicate lysis in target cells and a second condition selected to indicate lysis in other tissues or biological fluids (e.g., blood or serum). The evaluation can be performed in a cell-free system, cells, cell cultures, organ or tissue cultures, or whole animals. Preliminary evaluation under cell-free or culture conditions and confirmation by further evaluation of the whole animal may be useful. In some embodiments, useful candidate linkers lyse at least 2, 4, 10, 20, 50, 70, or 100 times faster in the target cells (or under in vitro conditions selected to mimic intracellular conditions) than blood or serum (or under in vitro conditions selected to mimic extracellular conditions).

In other embodiments, redox cleavable linkers are used. Redox cleavable linkers cleave upon reduction or oxidation. An example of a reducing cleavable group is a disulfide linkage group (-S-S-). To determine whether a candidate cleavable linker is a suitable "reducible cleavable linker," or, for example, suitable for use with a particular RNAi construct and a particular ligand, one or more of the methods described herein may be used. For example, candidate linkers can be evaluated by incubation with Dithiothreitol (DTT) or other reducing agents known in the art, which mimic the rate of lysis that would be observed in a cell (e.g., a target cell). Candidate linkers may also be evaluated under conditions selected to mimic blood or serum conditions. In certain embodiments, the candidate linker is cleaved in blood by at most 10%. In other embodiments, useful candidate linkers degrade at least 2-fold, 4-fold, 10-fold, 20-fold, 50-fold, 70-fold, or 100-fold faster in the cell (or under in vitro conditions selected to mimic intracellular conditions) than blood (or under in vitro conditions selected to mimic extracellular conditions).

In still other embodiments, the phosphate-based cleavable linker is cleaved by an agent that degrades or hydrolyzes the phosphate group. Examples of agents that hydrolyze phosphate groups in cells are enzymes, such as phosphatases in cells. -O-P (S) (SRk) -O-, O-and S-groups-S-P (O) (ORk) -O-, -O-P (S) (SRk) -O-, -S-P (O) (ORk) -O-, and-O-P (O) (ORk) -S-, -S-P (O) (ORk) -S-, S-and S-groups-O-P (S) (ORk) -S-, -S-P (S) (ORk) -O-, -O-P (O) (Rk) -O-, -O-P (S) (Rk) -O-, -S-P (O) (Rk) -O-, -S-P (S) (Rk) -O-, -S-P (O) (Rk) -S-, -O-P (S) (Rk) -S-. -S-P (O) (OH) -O- -O-P (O) (OH) -S-, -S-P (O) (OH) -O-, -O-P (O) (OH) -S-, and-S-P (O) (OH) -S-, -O-P (S) (OH) -S-, -SP (S) (OH) -O-, -O-P (O) (H) -O-, -O-P (S) (H) -O-, -S-P (O) (H) -O-, -S-P (S) (H) -O-, -S-P (O) (H) -S-, -O-P (S) (H) -S-. Another specific embodiment is-O-P (O) (OH) -O-. These candidate linkers can be evaluated using methods similar to those described above.

In other embodiments, the linker may comprise acid cleavable groups, which are groups that cleave under acidic conditions. In some embodiments, the acid cleavable group is cleaved in an acidic environment at a pH of about 6.5 or less (e.g., about 6.0, 5.5, 5.0 or less), or by a reagent such as an enzyme that can act as a general acid. In cells, specific low pH organelles, such as endosomes and lysosomes, can provide a cleavage environment for acid cleavable groups. Examples of acid cleavable linking groups include, but are not limited to, hydrazones, esters, and amino acid esters. The acid cleavable group may have the general formula-c=nn-, C (O) O or-OC (O). A particular embodiment is when the carbon attached to the oxygen of the ester (alkoxy) is aryl, substituted alkyl or tertiary alkyl such as dimethyl, pentyl or tertiary butyl. These candidates can be evaluated using methods similar to those described above.

In other embodiments, the linker may comprise ester-based cleavable groups that are cleaved by enzymes in the cell, such as esterases and amidases. Examples of ester-based cleavable groups include, but are not limited to, esters of alkylene, alkenylene, and alkynylene. The ester cleavable group has the general formula-C (O) O-or-OC (O) -. These candidate linkers can be evaluated using methods similar to those described above.

In other embodiments, the linker may comprise peptide-based cleavable groups that are cleaved by enzymes in the cell, such as peptidases and proteases. Peptide-based cleavable groups are peptide bonds formed between amino acids to produce oligopeptides (e.g., dipeptides, tripeptides, etc.) and polypeptides. The peptide-based cleavable group does not include an amide group (-C (O) NH-). The amido group may be formed between any alkylene, alkenylene or alkynylene group. Peptide bonds are a special type of amide bond formed between amino acids to produce peptides and proteins. Peptide-based cleavage groups are generally limited to peptide bonds (i.e., amide bonds) formed between the peptide-producing amino acid and the protein, and do not include the entire amide functionality. The peptide-based cleavable linker has the general formula-NHCHRAC (O) NHCHRBC (O) -, wherein RA and RB are R groups of two adjacent amino acids. These candidates can be evaluated using methods similar to those described above.

Other types of linkers suitable for attaching ligands to the sense strand or antisense strand in RNAi constructs of the invention are known in the art and can include linkers described in: U.S. patent No. 7,723,509;8,017,762;8,828,956;8,877,917; and 9,181,551, all of which are hereby incorporated by reference in their entirety.

In certain embodiments, the ligand covalently attached to the sense or antisense strand of the RNAi constructs of the invention comprises a GalNAc moiety, e.g., a multivalent GalNAc moiety. In some embodiments, the multivalent GalNAc moiety is a trivalent GalNAc moiety and is attached to the 3' end of the sense strand. In other embodiments, the multivalent GalNAc moiety is a trivalent GalNAc moiety and is attached to the 5' end of the sense strand. In still other embodiments, the multivalent GalNAc moiety is a tetravalent GalNAc moiety and is attached to the 3' end of the sense strand. In other embodiments, the multivalent GalNAc moiety is a tetravalent GalNAc moiety and is attached to the 5' end of the sense strand. In still other embodiments, the multivalent GalNAc moiety is a tetravalent GalNAc moiety and is attached to the 3' end of the sense strand. In other embodiments, the multivalent GalNAc moiety is a tetravalent GalNAc moiety and is attached to the 5' end of the sense strand. In some embodiments, the GalNAc moiety is attached to the 5' end of the sense strand of the odd numbered sequences SEQ ID NOS: 1-645 or 647-1291.

In some embodiments, RNAi constructs of the invention can be delivered to a target cell or tissue by administering a vector encoding and controlling intracellular expression of the RNAi construct. A "vector" (also referred to herein as an "expression vector") is a composition of matter that can be used to deliver a nucleic acid of interest into the interior of a cell. Many vectors are known in the art, including but not limited to linear polynucleotides, polynucleotides associated with ionic or amphiphilic compounds, plasmids, and viruses. Thus, the term "vector" includes autonomously replicating plasmids or viruses. Examples of viral vectors include, but are not limited to, adenovirus vectors, adeno-associated virus vectors, retrovirus vectors, and the like. The vector may replicate in living cells, or may be synthetically produced.

Typically, the vector used to express the RNAi constructs of the invention will comprise one or more promoters operably linked to the sequence encoding the RNAi construct. The phrase "operably linked" or "under transcriptional control" as used herein refers to a promoter that is in the correct position and orientation relative to a polynucleotide sequence to control transcription initiation by an RNA polymerase and expression of the polynucleotide sequence. "promoter" refers to a sequence recognized by a cellular synthesis mechanism or by an introduced synthesis mechanism that is required to initiate sequence-specific transcription of a gene. Suitable promoters include, but are not limited to, RNA pol I, pol II, HI or U6 RNA pol III, and viral promoters (e.g., the human Cytomegalovirus (CMV) immediate early gene promoter, the SV40 early promoter, and the Rous sarcoma virus long terminal repeat). In some embodiments, the HI or U6 RNA pol III promoter is preferred. The promoter may be a tissue-specific or inducible promoter. Of particular interest are liver-specific promoters, such as promoter sequences from the human α1-antitrypsin gene, albumin gene, heme binding protein gene, and liver lipase gene. Inducible promoters include those regulated by ecdysone, estrogen, progesterone, tetracycline, and isopropyl-PD 1-thiogalactoside (IPTG).

In some embodiments where the RNAi construct comprises siRNA, the two separate strands (the sense strand and the antisense strand) can be expressed from a single vector or two separate vectors. For example, in one embodiment, the sequence encoding the sense strand is operably linked to a promoter on a first vector and the sequence encoding the antisense strand is operably linked to a promoter on a second vector. In this embodiment, the first vector and the second vector are introduced into the target cell together, such as by infection or transfection, such that once transcribed, the sense strand and the antisense strand will hybridize within the cell to form the siRNA molecule. In another embodiment, the sense strand and the antisense strand are transcribed from two separate promoters located in a single vector. In some such embodiments, the sequence encoding the sense strand is operably linked to a first promoter and the sequence encoding the antisense strand is operably linked to a second promoter, wherein the first promoter and the second promoter are in a single vector. In one embodiment, the vector comprises a first promoter operably linked to a sequence encoding an siRNA molecule and a second promoter operably linked to the same sequence in the opposite direction such that transcription of the sequence from the first promoter results in synthesis of the sense strand of the siRNA molecule and transcription of the sequence from the second promoter results in synthesis of the antisense strand of the siRNA molecule.

In other embodiments where the RNAi construct comprises shRNA, the sequence encoding a single at least partially self-complementary RNA molecule is operably linked to a promoter to produce a single transcript. In some embodiments, the sequence encoding the shRNA comprises inverted repeats linked by a linker polynucleotide sequence to produce, upon transcription, the stem and loop structure of the shRNA.

In some embodiments, the vector encoding the RNAi constructs of the invention is a viral vector. Various viral vector systems suitable for expressing the RNAi constructs described herein include, but are not limited to, adenovirus vectors, retrovirus vectors (e.g., lentiviral vectors, moloney murine leukemia virus), adeno-associated virus vectors; herpes simplex virus vectors; SV 40 vector; polyoma viral vectors; papilloma virus vectors; a picornaviral vector; and poxvirus vectors (e.g., vaccinia virus). In certain embodiments, the viral vector is a retroviral vector (e.g., a lentiviral vector).

Various vectors suitable for use in the present invention, methods for inserting nucleic acid sequences encoding siRNA or shRNA molecules into vectors, and methods for delivering vectors to target cells are well within the ability of one of ordinary skill in the art. See, e.g., dornburg, gene therapy [ Gene therapy ], vol.2:301-310, 1995; eglitis, biotechnology [ Biotechnology ], volume 6:608-614, 1988; miller, humGENE therapeutic [ human Gene therapy ], volume 1:5-14,1990; anderson, nature, volume 392:25-30,1998; rubinson D A et al, nat. Genet. [ Nature genet ], volume 33:401-406, 2003; brummelkamp et al Science [ Science ], volume 296:550-553, 2002; brummelkamp et al, cancer Cell, volume 2:243-247, 2002; lee et al, nat Biotechnol Nature Biotechnology, vol.20:500-505, 2002; miyagishi et al, nat Biotechnol [ Nature Biotechnology ], vol.20:497-500, 2002; paddison et al, general Dev [ Gene development ], vol 16:948-958, 2002; paul et al, nat Biotechnol [ Nature Biotechnology ], vol.20:505-508, 2002; sui et al, procNatl Acad Sci USA [ Proc. Natl. Acad. Sci. USA ], volume 99:5515-5520, 2002; and Yu et al, proc Natl Acad Sci USA [ Proc. Natl. Acad. Sci. USA ], vol. 99:6047-6052, 2002, all of which are incorporated herein by reference in their entirety.

The invention also includes pharmaceutical compositions and formulations comprising the RNAi constructs described herein and a pharmaceutically acceptable carrier, excipient, or diluent. Such compositions and formulations are useful for reducing expression of HSD17B13 in a subject in need thereof. When clinical use is contemplated, the pharmaceutical compositions and formulations will be prepared in a form suitable for the intended use. In general, this will require the preparation of a composition that is substantially free of pyrogens and other impurities that may be harmful to humans or animals.

The phrase "pharmaceutically acceptable" or "pharmacologically acceptable" refers to molecular entities and compositions that do not produce adverse, allergic, or other untoward reactions when administered to an animal or human. As used herein, "pharmaceutically acceptable carrier, excipient or diluent" includes solvents, buffers, solutions, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like, which are acceptable for use in formulating medicaments, such as medicaments suitable for administration to humans. The use of such media and agents for pharmaceutically active substances is well known in the art. Unless any conventional medium or agent is incompatible with the RNAi constructs of the invention, its use in therapeutic compositions is contemplated. Supplementary active ingredients may also be incorporated into the compositions provided that they do not inactivate the carrier or RNAi construct of the composition.

The compositions and methods used to formulate the pharmaceutical compositions depend on a number of criteria including, but not limited to, the route of administration, the type and extent of the disease or disorder to be treated, or the dosage administered. In some embodiments, the pharmaceutical composition is formulated based on the intended route of delivery. For example, in certain embodiments, the pharmaceutical composition is formulated for parenteral delivery. Parenteral delivery forms include intravenous, intra-arterial, subcutaneous, intrathecal, intraperitoneal or intramuscular injection or infusion. In one embodiment, the pharmaceutical composition is formulated for intravenous delivery. In this embodiment, the pharmaceutical composition may comprise a lipid-based delivery vehicle. In another embodiment, the pharmaceutical composition is formulated for subcutaneous delivery. In this embodiment, the pharmaceutical composition can include a targeting ligand (e.g., a GalNAc-containing ligand as described herein).

In some embodiments, the pharmaceutical composition comprises an effective amount of an RNAi construct described herein. An "effective amount" is an amount sufficient to produce a beneficial or desired clinical result. In some embodiments, the effective amount is an amount sufficient to reduce HSD17B13 expression in hepatocytes of the subject. In some embodiments, the effective amount may be an amount sufficient to only partially reduce expression of HSD17B13, e.g., to a level comparable to expression of a wild-type HSD17B13 allele in a human heterozygote.

An effective amount of an RNAi construct of the present invention may be from about 0.01mg/kg body weight to about 100mg/kg body weight, about 0.05mg/kg body weight to about 75mg/kg body weight, about 0.1mg/kg body weight to about 50mg/kg body weight, about 1mg/kg to about 30mg/kg body weight, about 2.5mg/kg body weight to about 20mg/kg body weight, or about 5mg/kg body weight to about 15mg/kg body weight. In certain embodiments, a single effective dose of an RNAi construct of the present invention may be about 0.1mg/kg, about 0.5mg/kg, about 1mg/kg, about 2mg/kg, about 3mg/kg, about 4mg/kg, about 5mg/kg, about 6mg/kg, about 7mg/kg, about 8mg/kg, about 9mg/kg, or about 10mg/kg. Pharmaceutical compositions comprising an effective amount of an RNAi construct can be administered weekly, biweekly, monthly, quarterly, or half a year. Accurate determination of effective amounts and frequency of administration can be based on several factors, including patient size, age and general condition, the type of disorder to be treated (e.g., myocardial infarction, heart failure, coronary artery disease, hypercholesterolemia), the particular RNAi construct used, and the route of administration. Estimates of effective dosages and in vivo half-lives of any particular RNAi constructs of the invention can be determined using conventional methods and/or testing in a suitable animal model.

Administration of the pharmaceutical composition of the present invention may be via any conventional route, as long as the target tissue is obtainable via that route. Such approaches include, but are not limited to: parenteral (e.g., subcutaneous, intramuscular, intraperitoneal, or intravenous), buccal, nasal, buccal, intradermal, transdermal, and sublingual routes, or by injection directly into liver tissue or through the portal vein. In some embodiments, the pharmaceutical composition is administered parenterally. For example, in certain embodiments, the pharmaceutical composition is administered intravenously. In other embodiments, the pharmaceutical composition is administered subcutaneously.

Colloidal dispersion systems, such as macromolecular complexes, nanocapsules, microspheres, beads, and lipid-based systems, including oil-in-water emulsions, micelles, mixed micelles, and liposomes, can be used as delivery vehicles for RNAi constructs of the invention or vectors encoding such structures. Commercially available fat emulsions suitable for delivering the nucleic acids of the present invention include

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III, nutrilipid, and other similar lipid emulsions. The preferred colloidal system for use as an in vivo delivery vehicle is a liposome (i.e., an artificial membrane vesicle). The RNAi constructs of the invention can be encapsulated within liposomes or can form complexes therewith, particularly with cationic liposomes. Alternatively, the RNAi constructs of the invention can be complexed with lipids, particularly with cationic lipids. Suitable lipids and liposomes include neutral (e.g., dioleoyl phosphatidylethanolamine (DOPE), dimyristoyl phosphatidylcholine (DMPC), and dipalmitoyl phosphatidylcholine (DPPC)), distearoyl phosphatidylcholine, and negative (e.g., dimyristoyl phosphatidylglycerol (DMPG), and cationic (e.g., dioleoyl tetra -yl aminopropyl (DOTAP), and dioleoyl phosphatidylethanolamine (DOTMA)). The preparation and use of such colloidal dispersion systems is well known in the art. Exemplary formulations are also disclosed in U.S. Pat. nos. 5,981,505; U.S. patent No. 6,217,900; U.S. patent No. 6,383,512; U.S. patent No. 5,783,565; U.S. Pat. nos. 7,202,227; U.S. Pat. nos. 6,379,965; U.S. Pat. nos. 6,127,170; U.S. patent No. 5,837,533; U.S. patent No. 6,747,014; and W0.03/093449.

In some embodiments, RNAi constructs of the invention are fully encapsulated in a lipid formulation, e.g., to form SPLP, pSPLP, SNALP, or other nucleic acid-lipid particles. As used herein, the term "SNALP" refers to stable nucleic acid-lipid particles, including SPLP. As used herein, the term "SPLP" refers to a nucleic acid-lipid particle comprising plasmid DNA encapsulated within lipid vesicles. SNALP and SPLP typically contain cationic lipids, non-cationic lipids, and lipids that prevent aggregation of the particles (e.g., PEG-lipid conjugates). SNALP and SPLP are particularly useful for systemic applications because they exhibit extended cycle life following intravenous injection and accumulate at remote sites (e.g., sites physically separated from the site of administration). SPLP includes "pSPLP", which includes the encapsulated condensing agent-nucleic acid complex described in PCT publication No. WO 00/03683. The nucleic acid-lipid particles typically have an average diameter of about 50nm to about 150nm, about 60nm to about 130nm, about 70nm to about 110nm, or about 70nm to about 90nm, and are substantially non-toxic. In addition, the nucleic acid, when present in the nucleic acid-lipid particle, is resistant to degradation with nucleases in aqueous solution. Nucleic acid-lipid particles and methods of making the same are disclosed, for example, in U.S. patent No. 5,976,567;5,981,501;6,534,484;6,586,410;6,815,432; and PCT publication number WO 96/40964.

Pharmaceutical compositions suitable for injectable use include, for example, sterile aqueous solutions or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersions. Typically, these formulations are sterile and fluid to the extent that easy injection is possible. The formulations should remain stable under the conditions of manufacture and storage and should be protected from the contaminating action of microorganisms such as bacteria and fungi. Suitable solvents or dispersion media may contain, for example, water, ethanol, polyols (e.g., glycerol, propylene glycol, and liquid polyethylene glycols, and the like), suitable mixtures thereof, and vegetable oils. Proper fluidity can be maintained, for example, by the use of a cloth such as lecithin, by the maintenance of the required particle size in the case of dispersions and by the use of surfactants. The action of microorganisms can be prevented by various antibacterial antifungal agents, such as parabens, chlorobutanol, phenol, sorbic acid, thimerosal, and the like. In many cases, it is preferable to include isotonic agents, for example, sugars or sodium chloride. Prolonged absorption of the injectable compositions can be brought about by the use in the composition of absorption delaying agents, for example, aluminum monostearate and gelatin.

Sterile injectable solutions may be prepared by incorporating the active compound in the appropriate amount in the solvent with any other ingredients, e.g., as enumerated above, as required, followed by filtered sterilization. Generally, dispersions are prepared by incorporating the various sterilized active ingredients into a sterile vehicle which contains the basic dispersion medium and the required other ingredients, for example as enumerated above. In the case of sterile powders for the preparation of sterile injectable solutions, the preferred methods of preparation include vacuum-drying and freeze-drying techniques which yield a powder of the active ingredient(s) plus any additional desired ingredient from a previously sterile-filtered solution thereof.

The compositions of the present invention may generally be formulated in neutral or salt form. Pharmaceutically acceptable salts include, for example, acid addition salts (formed with free amino groups) derived from inorganic acids (e.g., hydrochloric or phosphoric acid) or from organic acids (e.g., acetic, oxalic, tartaric, mandelic, and the like). Salts formed with the free carboxyl groups may also be derived from inorganic bases (e.g., sodium, potassium, ammonium, calcium, or ferric hydroxides) or organic bases (e.g., isopropylamine, trimethylamine, histidine, procaine, and the like).

For parenteral administration in aqueous solution, for example, the solution is typically buffered appropriately and the liquid diluent is first rendered isotonic, for example with sufficient saline or glucose. Such aqueous solutions may be used, for example, for intravenous, intramuscular, subcutaneous and intraperitoneal administration. Preferably, a sterile aqueous medium is used, as known to one of ordinary skill in the art, particularly in light of the present disclosure. For example, a single dose may be dissolved in 1ml of isotonic NaCl solution and added to 1000ml of subcutaneous injection or injected at the proposed infusion site (see, e.g., "Remington's Pharmaceutical Sciences [ Lemington pharmaceutical science ]", 15 th edition, pages 1035-1038 and 1570-1580). For human administration, the formulation should meet sterility, pyrogenicity, general safety and purity standards as required by FDA standards. In certain embodiments, the pharmaceutical compositions of the invention comprise or consist of a sterile saline solution and an RNAi construct described herein. In other embodiments, the pharmaceutical compositions of the invention comprise or consist of an RNAi construct described herein and sterile water (e.g., water for injection, WFI). In still other embodiments, the pharmaceutical compositions of the invention comprise or consist of an RNAi construct described herein and phosphate-buffered saline (PBS).

In some embodiments, the pharmaceutical compositions of the present invention are packaged or stored within a device for administration. Devices for injectable formulations include, but are not limited to, injection ports, pre-filled syringes, auto-injectors, syringe pumps, in-body syringes, and injection pens. Devices for aerosolizing or powder formulations include, but are not limited to, inhalers, insufflators, inhalers, and the like. Thus, the present invention includes an administration device comprising a pharmaceutical composition of the present invention for use in the treatment or prevention of one or more disorders described herein.

Methods for inhibiting HSD17B13 expression

The invention also provides methods of inhibiting the expression of the HSD17B13 gene in a cell. These methods comprise contacting the cell with an RNAi construct (e.g., a double-stranded RNAi construct) in an amount effective to inhibit expression of HSD17B13 in the cell, thereby inhibiting expression of HSD17B13 in the cell. The contacting of the cells with the RNAi construct (e.g., double stranded RNAi construct) can be performed in vitro or in vivo. Contacting the cells with the RNAi construct in vivo includes contacting the cells or cell populations within a subject (e.g., a human subject) with the RNAi construct. Combinations of methods of contacting cells in vitro and in vivo are also possible.

The present invention provides methods for reducing or inhibiting HSD17B13 expression in a subject in need thereof and methods of treating or preventing a condition, disease or disorder associated with HSD17B13 expression or activity. By "condition, disease or disorder associated with HSD17B13 expression" is meant a condition, disease or disorder in which altered levels of HSD17B13 expression or elevated levels of HSD17B13 expression are associated with an increased risk of developing the condition, disease or disorder.

As described above, the contacting cells may be direct or indirect. Furthermore, contact with the cells may be achieved via targeting ligands, including any of the ligands described herein or known in the art. In preferred embodiments, the targeting ligand is a carbohydrate moiety, e.g., a GalNAc ligand, or a trivalent GalNAc moiety, or any other ligand that directs the RNAi construct to the site of interest.

In one embodiment, contacting the cell with the RNAi construct comprises "introducing" or "delivering" the RNAi construct into the cell by promoting or affecting uptake or uptake by the cell. Uptake or uptake of the RNAi construct can occur by independent diffusion or active cellular processes, or by the use of adjuvants or devices. The introduction of the RNAi construct into the cell can be performed in vitro and/or in vivo. For example, for in vivo introduction, the RNAi construct may be injected into the tissue site or administered systemically. In vitro introduction into cells includes methods known in the art, such as electroporation and lipofection. Other methods are described below and/or are known in the art.

As used herein, the term "inhibit" is used interchangeably with "reduce," "silence," "down-regulate," "repression," and other similar terms, and includes any level of inhibition.

The phrase "inhibiting HSD17B13 expression" is intended to mean inhibiting the expression of any HSD17B13 gene (e.g., a mouse HSD17B13 gene, a rat HSD17B13 gene, a monkey HSD17B13 gene, or a human HSD17B13 gene) as well as variants or mutants of the HSD17B13 gene. Thus, in the context of a genetically manipulated cell, cell population, or organism, the HSD17B13 gene may be a wild-type HSD17B13 gene, a mutant HSD17B13 gene, or a transgenic HSD17B13 gene.

"inhibiting expression of the HSD17B13 gene" includes any level of HSD17B13 gene inhibition, e.g., at least partial repression of the expression of the HSD17B13 gene. Expression of the HSD17B13 gene may be assessed based on the level or change in the level of any variable associated with HSD17B13 gene expression, such as HSD17B13 mRNA level, HSD17B13 protein level, or the amount or extent of amyloid deposits. The level can be assessed in a single cell or cell population, including, for example, a sample derived from a subject.

Inhibition may be assessed by a decrease in the absolute or relative level of one or more variables associated with HSD17B13 expression as compared to a control level. The control level may be any type of control level used in the art, e.g., a pre-dosing baseline level, or a level determined from a similar subject, cell, or sample that is untreated or treated with a control (e.g., a buffer-only control or a non-active agent control). In some embodiments of the methods of the invention, the expression of the HSD17B13 gene is inhibited by at least about 5%, at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99%.

Inhibition of HSD17B13 gene expression may be manifested by a reduction in the amount of mRNA expressed by a first cell or population of cells (such cells may be present in, for example, a sample derived from a subject) in which HSD17B13 is transcribed and has been treated (e.g., by contacting one or more cells with an RNAi construct of the invention, or by administering an RNAi construct of the invention to a subject in which such cells are present or have been present) such that expression of the HSD17B13 gene is inhibited compared to a second cell or population of cells (control cells) that is substantially identical to the first cell or population of cells but has not been so treated. In a preferred embodiment, inhibition is assessed by expressing mRNA levels in the treated cells as a percentage of mRNA levels in the control cells using the formula:

alternatively, inhibition of HSD17B13 gene expression may be assessed according to a decrease in a parameter functionally related to HSD17B13 gene expression. HSD17B13 gene silencing may be constitutive in any cell expressing HSD17B13 or by genomic engineering and determined by any assay known in the art.

Inhibition of HSD17B13 protein expression may be demonstrated by a decrease in the level of HSD17B13 protein expressed by a cell or cell population (e.g., the level of protein expressed in a sample derived from a subject). As described above, to assess mRNA repression, inhibition of protein expression levels in a treated cell or cell population can be similarly expressed as a percentage of protein levels in a control cell or cell population.

Control cells or cell populations useful for assessing inhibition of HSD17B13 gene expression include cells or cell populations that have not been contacted with the RNAi constructs of the invention. For example, the control cell or cell population can be derived from a separate subject (e.g., a human or animal subject) prior to treatment of the subject with the RNAi construct.

The level of HSD17B13 mRNA expressed by a cell or cell population or the level of circulating HSD17B13 mRNA may be determined using any method known in the art for assessing mRNA expression. In one embodiment, the level of HSD17B13 expression in the sample is determined by detecting mRNA of the transcribed polynucleotide, or a portion thereof, e.g., the HSD17B13 gene. RNA can be extracted from cells using RNA extraction techniques, including, for example, using acidic phenol/guanidine isothiocyanate extraction (RNAzol B; biogenesis), RNeasy RNA preparation kit (Qiagen), or PAXgene (PreAnalytix, switzerland). Typical assay formats utilizing ribonucleic acid hybridization include nuclear continuous assays, RT-PCR, RNase protection assays (Melton et al, nuc. Acids Res. [ nucleic acids Ind. ] 12:7035), northern blotting, in situ hybridization, and microarray analysis. The circulating mRNA can be detected using the method described in PCT/US 2012/043584, which is hereby incorporated by reference.

In one embodiment, the HSD17B13 expression level is determined using a nucleic acid probe. As used herein, the term "probe" refers to any molecule capable of selectively binding to a particular HSD17B 13. Probes may be synthesized by one of ordinary skill in the art or derived from an appropriate biological agent. Probes may be specifically designed for labeling. Examples of molecules that can be used as probes include, but are not limited to, RNA, DNA, proteins, antibodies, and organic molecules.

The isolated mRNA can be used in hybridization or amplification assays including, but not limited to, southern or northern blot analysis, polymerase Chain Reaction (PCR) analysis, and probe arrays. One method for determining mRNA levels includes contacting the isolated mRNA with a nucleic acid molecule (probe) that hybridizes to HSD17B13 mRNA. In one embodiment, mRNA is immobilized on a solid surface and contacted with a probe, for example by running the isolated mRNA on an agarose gel and transferring the mRNA from the gel to a membrane, such as nitrocellulose. In an alternative embodiment, one or more probes are immobilized on a solid surface and mRNA is contacted with the one or more probes, for example in an Affymetrix gene chip array. The skilled artisan can readily employ known mRNA detection methods suitable for determining HSD17B13 mRNA levels.

Alternative methods for determining the expression level of HSD17B13 in a sample include procedures such as nucleic acid amplification and/or reverse transcriptase (to prepare cDNA) of mRNA in the sample, for example by RT-PCR (experimental examples are set forth in Mullis,1987, U.S. Pat. No. 4,683,202), ligase chain reaction (Barany (1991) Proc.Natl. Acad. Sci. USA [ Proc.Natl.Acad.Sci.Sci.U.S. USA. Natl.A.)]88:189-193), self-sustained sequence replication (Guatelli et al (1990) Proc. Natl. Acad. Sci. USA [ Proc. Natl. Acad. Sci. USA, U.S. national academy of sciences)]87:1874-1878), a transcription amplification system (Kwoh et al (1989) Proc.Natl. Acad.Sci.USA [ Proc. Natl. Acad. Sci. USA, national academy of sciences USA ]]86:1173-1177), Q-beta replicase (Lizardi et al (1988) Bio/Technology [ Biotechnology ]]6:1197), rolling circle replication (Lizardi et al, U.S. Pat. No. 5,854,033) or any other nucleic acid amplification method, and then detecting the amplified molecules using techniques well known to those of ordinary skill in the art. These detection schemes are particularly useful for detecting nucleic acid molecules if these molecules are present in very low amounts. In a particular aspect of the invention, the detection of the target sequence is performed by fluorescent quantitative RT-PCR (i.e., taqMan ^TM System) to determine the expression level of HSD17B 13. The expression level of HSD17B13 mRNA can be monitored using membrane blotting (e.g., for hybridization analysis, e.g., northern blotting, southern blotting, spotting, etc.) or microwells, sample tubes, gels, beads, or fibers (or any solid support comprising bound nucleic acid). See U.S. patent U.S. Pat. Nos. 5,770,722, 5,874,219, 5,744,305, 5,677,195, and 5,445,934, which are incorporated herein by reference. Determination of HSD17B13 expression levels may also include the use of nucleic acid probes in solution.

In preferred embodiments, mRNA expression levels are assessed, for example, using branched DNA (bDNA) assays, real-time PCR (qPCR), or quantitative FISH assays. The uses of these methods are described and illustrated in the examples provided herein.

The HSD17B13 protein expression level may be determined using any method known in the art for measuring protein levels. Such methods include, for example, electrophoresis, capillary electrophoresis, high Performance Liquid Chromatography (HPLC), thin Layer Chromatography (TLC), super-diffusion chromatography, fluid or gel-precipitate reactions, absorption spectroscopy, colorimetric assays, spectrophotometric assays, flow cytometry, immunodiffusion (single or double), immunoelectrophoresis, immunoblotting, radioimmunoassay (RIA), enzyme-linked immunosorbent assay (ELISA), immunofluorescent assays, electrochemiluminescent assays, and the like.

In some embodiments, the efficacy of the methods of the invention may be monitored by detecting or monitoring the reduction in symptoms of HSD17B13 disease (e.g., biomarkers of liver disease, such as AST and ALT). These symptoms can be assessed in vitro or in vivo using any method known in the art.

In some embodiments of the methods of the invention, the RNAi construct is administered to the subject, thereby delivering the RNAi construct to a specific site within the subject. Inhibition of HSD17B13 expression can be assessed using measurements of HSD17B13 mRNA or HSD17B13 protein levels or changes in levels in a sample of fluid or tissue from a specific site within a subject. In a preferred embodiment, these sites are selected from the group consisting of liver, choroid plexus, retina, and pancreas. The site may also be a small portion or subset of cells from any of the above sites. The site may also include cells that express a particular type of receptor.

Methods for treating or preventing HSD17B 13-related disorders

The present invention provides methods of treatment and prophylaxis comprising administering a composition comprising RNAi, or a pharmaceutical composition comprising an RNAi construct, or a vector comprising an RNAi construct of the invention, to a subject suffering from, or susceptible to developing, an HSD17B 13-related disease, disorder and/or condition. Non-limiting examples of HSD17B 13-related diseases include, for example, fatty liver (steatosis), nonalcoholic steatohepatitis (NASH), cirrhosis, fat accumulation in the liver, liver inflammation, hepatocyte necrosis, liver fibrosis, obesity, or nonalcoholic fatty liver disease (NAFLD). In one embodiment, the HSD17B 13-related disorder is NAFLD. In another embodiment, the HSD17B 13-associated disorder is NASH. In another embodiment, the HSD17B 13-associated disorder is fatty liver (steatosis). In another embodiment, the HSD17B 13-associated disorder is insulin resistance. In another embodiment, the HSD17B 13-related disorder is not insulin resistance.

In certain embodiments, the invention provides a method for reducing HSD17B13 expression in a patient in need thereof, comprising administering to the patient any of the RNAi constructs described herein. As used herein, the term "patient" refers to mammals, including humans, and is used interchangeably with the term "subject". Preferably, the level of HSD17B13 expression in hepatocytes of the patient is reduced after administration of the RNAi construct compared to the level of HSD17B13 expression in a patient not receiving the RNAi construct.

The methods of the invention are useful for treating subjects suffering from HSD17B 13-related diseases, e.g., subjects who would benefit from reduced HSD17B13 gene expression and/or HSD17B13 protein production. In one aspect, the invention provides methods of reducing the level of 17β -hydroxysteroid dehydrogenase type 13 (HSD 17B 13) gene expression in a subject having non-alcoholic fatty liver disease (NAFLD). In another aspect, the invention provides a method of reducing the level of HSD17B13 protein in a subject having NAFLD.

In another aspect, the invention provides a method of treating a subject having NAFLD. In one aspect, the invention provides methods of treating a subject suffering from HSD17B 13-related disorders, such as fatty liver (steatosis), nonalcoholic steatohepatitis (NASH), cirrhosis, fat accumulation in the liver, liver inflammation, hepatocyte necrosis, liver fibrosis, obesity, or nonalcoholic fatty liver disease (NAFLD). The methods of treatment (and uses) of the invention comprise administering to a subject, e.g., a human, a therapeutically effective amount of an RNAi construct of the invention targeting the HSD17B13 gene, or a pharmaceutical composition comprising an RNAi construct of the invention targeting the HSD17B13 gene, or a vector of the invention comprising an RNAi construct targeting the HSD17B13 gene.

In one aspect, the invention provides methods of preventing at least one symptom, such as the presence of hedgehog signaling pathway elevation, fatigue, weakness, weight loss, loss of appetite, nausea, abdominal pain, spider-like vessels, yellowing of skin and eyes (jaundice), itching, leg fluid accumulation and swelling (edema), abdominal swelling (ascites), and confusion in a subject suffering from NAFLD. These methods comprise administering to a subject a therapeutically effective amount of an RNAi construct, e.g., a dsRNA, pharmaceutical composition, or vector of the invention, thereby preventing at least one symptom in a subject suffering from a disorder that would benefit from reduced HSD17B13 gene expression.

In another aspect, the invention provides the use of a therapeutically effective amount of an RNAi construct of the invention for treating a subject (e.g., a subject who would benefit from reduction and/or inhibition of HSD17B13 gene expression). In another aspect, the invention provides the use of an RNAi construct of the invention (e.g., dsRNA) targeting the HSD17B13 gene or a pharmaceutical composition comprising an RNAi construct targeting the HSB17B13 gene in the manufacture of a medicament for treating a subject, e.g., a subject who would benefit from reduced and/or inhibited HSD17B13 gene expression and/or HSD17B13 protein production, e.g., a subject suffering from a disorder that would benefit from reduced HSD17B13 protein expression (e.g., HSD17B 13-related disease).

In another aspect, the invention provides the use of RNAi (e.g., dsRNA) of the invention for preventing at least one symptom in a subject suffering from a disorder that would benefit from reduced and/or inhibited HSD17B13 gene expression and/or HSD17B13 protein production.

In another aspect, the invention provides the use of an RNAi construct of the invention in the manufacture of a medicament for preventing at least one symptom in a subject suffering from a disorder that would benefit from reduced and/or inhibited HSD17B13 gene expression and/or HSD17B13 protein production, e.g., a HSD17B 13-related disease.

In one embodiment, an RNAi construct targeting HSD17B13 is administered to a subject having a HSD17B 13-associated disease (e.g., non-alcoholic fatty liver disease (NAFLD)), such that, for example, expression of the HSD17B13 gene in a cell, tissue, blood, or other tissue or body fluid of the subject is reduced by at least about 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, 30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 86%, 88%, 92%, 90%, 91%, or more, 98%, or more, 93%, or more, when dsRNA agent is administered to the subject.

Methods and uses of the invention include administering the compositions described herein such that expression of the target HSD17B13 gene is reduced, e.g., for about 1, 2, 3, 4, 5, 6, 7, 8, 12, 16, 18, 24, 28, 32, 36, 40, 44, 48, 52, 56, 60, 64, 68, 72, 76, or about 80 hours. In one embodiment, the reduced expression of the target HSD17B13 gene is for an extended period of time, e.g., at least about two, three, four, five, six, seven days or more, e.g., about one week, two weeks, three weeks, or about four weeks or more.

Administration of dsRNA according to the methods and uses of the invention may result in a reduction in the severity, sign, symptom and/or marker of HSD17B 13-related diseases, such as non-alcoholic fatty liver disease (NAFLD), in patients suffering from such diseases or disorders. In this case, "reduced" refers to a statistically significant reduction in this level. The decrease may be, for example, at least about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or about 100%. The therapeutic or prophylactic efficacy of a disease can be assessed, for example, by measuring the level of disease progression, disease remission, symptom severity, pain relief, quality of life, the dosage of drug required to maintain therapeutic effect, disease markers, or any other measurable parameter suitable for the particular disease being treated or targeted for prophylaxis. It is within the ability of one of ordinary skill in the art to monitor therapeutic or prophylactic efficacy by measuring any one or any combination of these parameters. For example, NAFLD therapeutic efficacy can be assessed, for example, by periodic monitoring of NAFLD symptoms, liver fat levels, or expression of downstream genes. Comparison of the subsequent readings to the initial readings provides an indication to the physician as to whether the treatment is effective. It is within the ability of one of ordinary skill in the art to monitor therapeutic or prophylactic efficacy by measuring any one or any combination of these parameters. In combination with administration of RNAi constructs targeting HSD17B13 or pharmaceutical compositions thereof, an "effective against" HSD17B 13-related disease is indicative of at least beneficial effects on a statistically significant portion of the patient, such as improvement of symptoms, cure, disease alleviation, life prolongation, quality of life improvement, or other effects commonly recognized as positive by physicians familiar with the treatment of NAFLD and/or HSD17B 13-related disease and related etiology, administered in a clinically appropriate manner.

The therapeutic or prophylactic effect is evident when one or more parameters of the disease state are statistically significantly improved, or when symptoms are not worsened or otherwise expected to occur as a result. For example, a favorable change of at least 10%, preferably at least 20%, 30%, 40%, 50% or more in a measurable disease parameter may be indicative of an effective treatment. Experimental animal models known in the art for a given disease can also be used to determine the efficacy of a given RNAi drug or formulation of the drug. Treatment efficacy was demonstrated when statistically significant reduction in markers or symptoms was observed when experimental animal models were used.

Administration of the RNAi construct can reduce the presence of HSD17B13 protein levels (e.g., in a cell, tissue, blood, urine, or other compartment of a patient) by at least about 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, 30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or at least about 99% or more.

A smaller dose of the drug (e.g., 5% infusion) may be administered to the patient and adverse effects (e.g., allergic reactions) monitored prior to administration of the full dose of the RNAi construct. In another example, a patient may be monitored for unwanted immunostimulatory effects, such as increased cytokine (e.g., TNF- α or INF- α) levels.

The composition according to the present invention or the pharmaceutical composition prepared therefrom can improve the quality of life due to the inhibitory effect on HSD17B13 expression.

The RNAi constructs of the invention can be administered in "naked" form, wherein the modified or unmodified RNAi construct is suspended directly in an aqueous solvent or suitable buffer solvent in a "free RNAi" manner. Free RNAi is administered in the absence of the pharmaceutical composition.

Alternatively, RNAi of the invention can be administered as a pharmaceutical composition, e.g., dsRNA liposome formulation.

Subjects who benefit from reduced and/or inhibited HSD17B13 gene expression are those suffering from non-alcoholic fatty liver disease (NAFLD) and/or HSD17B13 related diseases or disorders, as described herein.

Treatment of a subject who would benefit from reduced and/or inhibited HSD17B13 gene expression includes therapeutic and prophylactic treatment.

The invention also provides methods and uses of RNAi constructs, or pharmaceutical compositions thereof, for use in combination with other drugs and/or other methods of treatment, e.g., with known drugs and/or known methods of treatment, e.g., such as those currently used to treat such disorders, to treat subjects, e.g., subjects suffering from HSD17B 13-related diseases, who would benefit from reduced and/or inhibited HSD17B13 gene expression.

For example, in certain embodiments, RNAi constructs targeting the HSD17B13 gene are administered in combination with an agent, e.g., as described elsewhere herein, for treating HSD17B 13-related diseases. For example, other therapeutic agents and methods of treatment suitable for treating a subject that would benefit from reduced expression of HSD17B13 (e.g., a subject having an HSD17B 13-related disease) include RNAi constructs, therapeutic agents, and/or procedures targeting different portions of the HSD17B13 gene, or a combination of any of the foregoing, for treating an HSD17B 13-related disease.

In certain embodiments, the first RNAi construct targeting the HSD17B13 gene is administered in combination with a second RNAi construct targeting a different portion of the HSD17B13 gene. For example, a first RNAi construct comprises a first sense strand and a first antisense strand forming a double-stranded region, wherein substantially all of the nucleotides of the first sense strand and substantially all of the nucleotides of the first antisense strand are modified nucleotides, wherein the first sense strand is conjugated to a ligand attached at the 3' -terminus, and wherein the ligand is one or more GalNAc derivatives attached via a divalent or trivalent branched linker; and the second RNAi construct comprises a second sense strand and a second antisense strand forming a double-stranded region, wherein substantially all of the nucleotides of the second sense strand and substantially all of the nucleotides of the second antisense strand are modified nucleotides, wherein the second sense strand is conjugated to a ligand attached at the 3' -end, and wherein the ligand is one or more GalNAc derivatives attached via a divalent or trivalent branched linker.

In one embodiment, all nucleotides of the first and second sense strands and/or all nucleotides of the first and second antisense strands comprise modifications.

In one embodiment, at least one of the modified nucleotides is selected from the group consisting of: 3' -terminal deoxythymine (dT) nucleotides, 2' -O-methyl modified nucleotides, 2' -fluoro modified nucleotides, locked nucleotides, unlocked nucleotides, conformationally restricted nucleotides, restricted ethyl nucleotides, abasic nucleotides, 2' -amino modified nucleotides, 2' -O-allyl modified nucleotides, 2' -C-alkyl modified nucleotides, 2' -hydroxy modified nucleotides, 2' -methoxyethyl modified nucleotides, 2' -0-alkyl modified nucleotides, morpholino nucleotides, phosphoramidates, non-natural bases comprising nucleotides, tetrahydropyran modified nucleotides, 1, 5-hexitol modified nucleotides, cyclohexenyl modified nucleotides, phosphorothioate group containing nucleotides, methylphosphonate group containing nucleotides, 5' -phosphate containing nucleotides, and 5' -phosphate mimetic containing nucleotides.

In certain embodiments, a first RNAi construct targeting the HSD17B13 gene is administered in combination with a second RNAi construct targeting a gene other than the HSD17B13 gene. For example, an RNAi construct targeting the HSD17B13 gene can be administered in combination with an RNAi construct targeting the SCAP gene. The first RNAi construct targeting the HSD17B13 gene and the second RNAi construct targeting a gene other than the HSD17B13 gene (e.g., a SCAP gene) can be administered as part of the same pharmaceutical composition. Alternatively, a first RNAi construct targeting the HSD17B13 gene and a second RNAi construct targeting a gene other than the HSD17B13 gene (e.g., a SCAP gene) can be administered as part of different pharmaceutical compositions.

The RNAi construct and the additional therapeutic agent and/or treatment can be administered at the same time and/or in the same combination, e.g., parenterally, or the additional therapeutic agent can be administered as part of a separate composition or at a separate time and/or using another method known in the art or described herein.

The invention also provides methods of reducing and/or inhibiting HSD17B13 expression in a cell using the RNAi constructs of the invention and/or compositions comprising the RNAi constructs of the invention. In other aspects, the invention provides RNAi constructs of the invention and/or compositions comprising the RNAi constructs of the invention for reducing and/or inhibiting HSD17B13 gene expression in a cell. In still other aspects, there is provided the use of an RNAi of the invention and/or a composition comprising an RNAi of the invention in the manufacture of a medicament for reducing and/or inhibiting HSD17B13 gene expression in a cell. In still other aspects, the invention provides RNAi of the invention and/or a composition comprising RNAi of the invention for use in reducing and/or inhibiting HSD17B13 protein production in a cell. In still other aspects, there is provided the use of an RNAi of the invention and/or a composition comprising an RNAi of the invention in the manufacture of a medicament for reducing and/or inhibiting HSD17B13 protein production in a cell. These methods and uses include contacting a cell with an RNAi construct (e.g., dsRNA) of the invention, and maintaining the cell for a time sufficient to obtain degradation of HSD17B13 gene mRNA transcript, thereby inhibiting HSD17B13 gene expression or inhibiting HSD17B13 protein production in the cell.

The reduction in gene expression may be assessed by any method known in the art. For example, the reduction in HSD17B13 expression may be determined by using routine methods of one of ordinary skill in the art, such as northern blotting, qRT-PCR, to determine the mRNA expression level of HSD17B 13; determining the protein level of HSD17B13 by using routine methods of one of ordinary skill in the art, such as immunoblotting, immunological techniques, flow cytometry methods, ELISA; and/or by determining the biological activity of HSD17B 13.

In the methods and uses of the invention, the cells may be contacted in vitro or in vivo, i.e., the cells may be in a subject.

Cells suitable for treatment using the methods of the invention may be any cell expressing the HSD17B13 gene, e.g., cells from a subject with NAFLD or cells comprising an expression vector comprising the HSD17B13 gene or a portion of the HSD17B13 gene. Cells suitable for use in the methods and uses of the invention may be mammalian cells, such as primate cells (e.g., human cells or non-human primate cells, e.g., monkey cells or chimpanzee cells), non-primate cells (e.g., bovine cells, porcine cells, camel cells, llama cells, equine cells, caprine cells, rabbit cells, ovine cells, hamster cells, guinea pig cells, feline cells, canine cells, rat cells, mouse cells, lion cells, tiger cells, bear cells, or buffalo cells), avian cells (e.g., duck cells or goose cells), or whale cells. In one embodiment, the cell is a human cell.

HSD17B13 gene expression may be inhibited in a cell by at least about 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, 30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or about 100%.

HSD17B13 protein production may be inhibited in a cell by at least about 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, 30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or about 100%.

The in vivo methods and uses of the invention may include administering to a subject a composition comprising an RNAi construct, wherein the RNAi construct comprises a nucleotide sequence complementary to at least a portion of the RNA transcript of the HSD17B13 gene of the mammal to be treated. When the organism to be treated is a human, the composition may be administered by any means known in the art including, but not limited to, subcutaneous, intravenous, oral, intraperitoneal, or parenteral routes, including intracranial (e.g., intraventricular, intraparenchymal, and intrathecal), intramuscular, transdermal, airway (aerosol), nasal, rectal, and topical (including buccal and sublingual) administration. In certain embodiments, the composition is administered by subcutaneous or intravenous infusion or injection. In one embodiment, the composition is administered by subcutaneous injection.

In some embodiments, administration is via a long-acting injection. Long-acting injections may release RNAi in a consistent manner over an extended period of time. Thus, prolonged injection may reduce the frequency of administration required to obtain a desired effect, e.g., a desired HSD17B13 inhibitory or therapeutic or prophylactic effect. Long-acting injections may also provide more consistent serum concentrations. The long-acting injection may include subcutaneous injection or intramuscular injection. In a preferred embodiment, the long-acting injection is subcutaneous injection.

In some embodiments, administration is by a pump. The pump may be an external pump or a surgically implanted pump. In certain embodiments, the pump is a subcutaneously implanted osmotic pump. In other embodiments, the pump is an infusion pump. Infusion pumps may be used for intravenous, subcutaneous, arterial or epidural infusion. In a preferred embodiment, the infusion pump is a subcutaneous infusion pump. In other embodiments, the pump is a surgically implanted pump that delivers the RNAi construct to the subject.

The mode of administration may be selected based on whether local or systemic treatment is desired and based on the area to be treated. The route of administration and the site of administration may be selected to enhance targeting.

In one aspect, the invention also provides methods for inhibiting HSD17B13 gene expression in a mammal (e.g., a human). The invention also provides a composition comprising an RNAi construct, e.g., dsRNA, targeting the HSD17B13 gene in a mammalian cell for inhibiting HSD17B13 gene expression in a mammal. In another aspect, the invention provides the use of RNAi (e.g., dsRNA) targeting the HSD17B13 gene in a mammalian cell in the manufacture of a medicament for inhibiting expression of the HSD17B13 gene in a mammal.

These methods and uses include administering to a mammal (e.g., a human) a composition comprising RNAi (e.g., dsRNA) targeting the HSD17B13 gene in mammalian cells, and maintaining the mammal for a time sufficient to obtain degradation of mRNA transcripts of the HSD17B13 gene, thereby inhibiting HSD17B13 gene expression in the mammal.

The reduction in gene expression may be assessed in a peripheral blood sample of a subject administered via RNAi by any method known in the art (e.g., qRT-PCR as described herein). The reduction in protein production can be assessed by any method known in the art and by the methods described herein (e.g., ELISA or immunoblotting). In one embodiment, the tissue sample is used as a tissue material for monitoring reduced expression of the HSD17B13 gene and/or protein. In another embodiment, blood samples are used as tissue material for monitoring reduced expression of HSD17B13 genes and/or proteins.

In one embodiment, in vivo validation of RISC-mediated target cleavage following RNAi construct administration is accomplished by performing 5' -RACE or modification of protocols as known in the art (Lasham A et al (2010) Nucleic Acid Res. [ Nucleic acids research ],38 (3) p-el 9) (Zimmermann et al (2006) Nature [ Nature ] 441:111-4).

It is understood that all ribonucleic acid sequences disclosed herein can be converted to deoxyribonucleic acid sequences by substitution of uracil bases in the sequence with thymine bases. Likewise, all of the deoxyribonucleic acid sequences disclosed herein can be converted to ribonucleic acid sequences by substitution of thymine bases in the sequence with uracil bases. The present invention includes deoxyribonucleic acid sequences, ribonucleic acid sequences, and sequences comprising a mixture of deoxyribonucleotides and ribonucleotides of all sequences disclosed herein.

Additionally, any of the nucleic acid sequences disclosed herein can be modified with any combination of chemical modifications. Those of ordinary skill in the art will readily appreciate that in some cases, designations describing modified polynucleotides such as "RNA" or "DNA" are arbitrary. For example, a polynucleotide comprising a nucleotide having a 2' -OH substituent on ribose and a thymine base may be described as a DNA molecule having a modified sugar (2 ' -OH for the natural 2' -H of DNA) or an RNA molecule having a modified base (thymine (methylated uracil) for the natural uracil of RNA).

Thus, the nucleic acid sequences provided herein (including but not limited to the nucleic acid sequences in the sequence listing) are intended to encompass nucleic acids containing any combination of natural or modified RNAs and/or DNAs, including but not limited to such nucleic acids having modified nucleobases. As another example and without limitation, a polynucleotide having the sequence "ATCGATCG" encompasses any polynucleotide having such a sequence, modified or unmodified, including, but not limited to, compounds comprising RNA bases, such as those having the sequence "aucghac", and those having some DNA bases and some RNA bases (e.g., "aucghag"), and polynucleotides having other modified bases (e.g., "atmecghac"), wherein meC represents a cytosine base comprising a methyl group at the 5-position.

The following examples, including the results of experiments performed and implementations, are provided for illustrative purposes only and should not be construed as limiting the scope of the appended claims.

Incorporated by reference

All publications, patents, and patent applications mentioned in this specification are herein incorporated by reference to the same extent as if each individual publication, patent, or patent application was specifically and individually indicated to be incorporated by reference. However, citation of references herein should not be construed as an admission that such references are prior art to the present invention. Where any definition or terminology provided in a reference, which is incorporated by reference, is different from the terms and discussions provided herein, the terms and definitions of the present invention prevail.

Equivalents (Eq.)

The previous written description is to be considered as sufficient to enable one of ordinary skill in the art to practice the invention. The foregoing description and examples detail certain preferred embodiments of the invention and describe the best mode contemplated by the inventors. It will be appreciated, however, that no matter how detailed the foregoing appears in text, the invention may be practiced in many ways and the invention should be interpreted in accordance with the appended claims and any equivalents thereof.

The following examples (including the experiments performed and the results achieved) are provided for illustrative purposes only and are not intended to be construed as limiting the invention.

All animal experiments described herein were approved by the committee for animal protection and use (IACUC) of the underwriter's, and were cared according to the guidelines for laboratory animal care and use (Guide for the Care and Use of Laboratory Animals) version 8 (national research committee (National Research Council (u.s.)). The laboratory animal care and use guideline update committee, the american laboratory animal institute, and the national academy of sciences press (2011) Guide for the care and use of laboratory animals [ guidelines for care and use of laboratory animals ], 8 th edition, national academy of sciences press, washington, d.c.. Mice were individually housed at 22 ℃ ±2 ℃ with 12 hour light exposure; in an air-conditioned room (0600-1800 hours) with a 12 hour dark period. Unless otherwise indicated, animals were given regular food (Envigo, 2920X; or otherwise specified) ad libitum and water (reverse osmosis purification) via an automated watering system. At the end, blood was collected by cardiac puncture under deep anesthesia, and then euthanasia was performed by a secondary physical method according to laboratory animal care evaluation and certification institute (AAALAC) guidelines.

Example 1: selection, design and Synthesis of modified HSD17B13 siRNA molecules

Bioinformatic analysis of human HSD17B13 transcripts (nm_ 178135.4 or nm_ 001136230.2) was used to identify, identify and select the best sequence for therapeutic siRNA molecules targeting 17 beta-hydroxysteroid dehydrogenase type 13 (HSD 17B 13). Table 1 shows sequences identified as having therapeutic properties. Among the various sequences, { INVAB } is inverted abasic, { INVDA } is inverted deoxyadenosine, GNA is a diol nucleic acid, dT is deoxythymidine, and dC is deoxycytosine.

TABLE 1 siRNA sequences against HSD17B13

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To increase the efficacy and in vivo stability of HSD17B13 siRNA sequences, chemical modifications were incorporated into HSD17B13 siRNA molecules. Specifically, 2 '-O-methyl and 2' -fluoro modifications of ribose are incorporated at specific positions within HSD17B13 siRNA. Phosphorothioate internucleotide linkages are also incorporated at the ends of antisense and/or sense sequences. The modifications of the sense and antisense sequences of each modified HSD17B13 siRNA are described in table 2 below. The nucleotide sequences in table 2 and elsewhere in this application are listed according to the following symbols: A. u, G, and c=corresponding ribonucleotides; dT = deoxythymidine; dA = deoxyadenosine; dc=deoxycytidine; dG = deoxyguanosine; invDT = inverted deoxythymidine; invDA = reverse deoxyadenosine; invDC = inverted deoxycytidine; invDG = inverted deoxyguanosine; a. u, g, and c=corresponding 2' -O-methyl ribonucleotides; af. Uf, gf, and Cf = corresponding 2' -deoxy-2 ' -fluoro ("2 ' -fluoro") ribonucleotides; ab = abasic; meO-i=2' -methoxyinosine; GNA = diol nucleic acid; sGNA = diol nucleic acid with 3' phosphorothioate; LNA = locked nucleic acid; the insertion of "s" in the sequence indicates that two adjacent nucleotides are linked by a phosphorothioate diester group (e.g., phosphorothioate internucleotide linkages). Unless otherwise indicated, all other nucleotides are linked by a 3'-5' phosphodiester group. Each siRNA compound in table 2 comprises a 21 base pair duplex region with a 2 nucleotide overhang at the 3' end of both strands, or a passivating species at one or both ends. The 5' end of the sense strand in each siRNA compound has been linked by a phosphorothioate or phosphodiester linkage to a GalNAc structure of formula I:

Wherein x=o or S.

TABLE 2 siRNA sequences for HSD17B13 with modifications

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Example 3: microdroplet digital PCR assay for siRNA to HSD17B13-rs738409 and HSD17B13-rs738409-rs738408

Human primary hepatocytes (Xenotech/Sekisui donor lot HC 3-38) were thawed in Optitthaw medium (Xenotech cat# K8000), centrifuged and resuspended in OptiPlate hepatocyte medium (Xenotech cat# K8200) after media aspiration, and plated in 96-well collagen-coated plates (Greiner cat# 655950). After 2-4 hours of incubation, the medium was removed and replaced with OptiCulture hepatocyte medium (Xenotech company catalog number K8300). GalNAc conjugated siRNA was delivered to cells by free uptake (no transfection reagent) at various concentrations up to 3.8uM 2-4 hours after addition of opticure medium. The cells were incubated at 37℃and 5% CO2 for 24-72 hours. Cells were then lysed with Qiagen RLT buffer (79216) +1% 2-mercaptoethanol (Sigma, M-3148) and lysates were stored at-20 ℃. RNA was purified using Qiagen QIACube HT instrument (9001793) and Qiagen RNeasy 96QIACube HT kit (74171) according to manufacturer's instructions. Samples were analyzed using the QIAxpert system (9002340). cDNA was synthesized from the RNA samples using a Applied Biosystems High Capacity cDNA Reverse Transcription kit (4368813) and the RNA concentration was input as a function of sample according to the manufacturer's instructions for the assembly reaction. Reverse transcription was performed on a BioRad tetrad thermocycler (model PTC-0240G) under the following conditions: the temperature is maintained indefinitely at 25℃for 10 minutes, 37℃for 120 minutes, 85℃for 5 minutes, and then (optionally) 4 ℃.

Microdroplet digital PCR (ddPCR) was performed using a QX200 AutoDG microdroplet digital PCR system of BioRad according to manufacturer's instructions. The reactions were assembled into Eppendorf transparent 96-well PCR plates (951020303) using BioRad ddPCR Supermix for Probes (1863010) and qPCR assays using fluorescent labeled HSD17B13 (idths. Pt.58.21464637, primer to probe ratio 3.6:1 and TBP (IDT) hs. Pt.53a.20105486, probe ratio 3.6:1) and rnase-free water (Ambion company, AM 9937). The final primer/probe concentrations were 900nM/250nM, respectively, and different cDNA concentrations were entered into the wells. Droplets were formed using a BioRad Auto DG droplet generator (1864101) provided with manufacturer recommended consumables (BioRad DG32 cartridge 1864108, bioRad tip 1864121, eppendorf blue 96-well PCR plate 951020362, bioRad droplet-generating oil 1864110 for probes, and BioRad droplet plate assembly). Droplets were amplified on a BioRad C1000 touch thermocycler (1851197) using the following conditions: enzyme activation at 95 ℃ for 10 min, denaturation at 94 ℃ for 30 sec, then annealing/extension at 60 ℃ for 1 min, 40 cycles using a 2 ℃/sec ramp rate, enzyme inactivation at 98 ℃ for 10 min, and then (optionally) infinite hold at 4 ℃. The samples were then read on a BioRad QX200 droplet reader that measures FAM/HEX signals correlated to HSD17B13 or TBP concentration. Data were analyzed using the QuantaSoft software package of BioRad. Samples were gated by channel (fluorescent label) to determine the concentration of each sample. Each sample was then expressed as a ratio of target gene (HSD 17B 13) concentration/housekeeping gene (TBP) concentration in order to control sample loading differences. The data was then imported Genedata Screener, where each test siRNA was normalized to the median of the neutral control wells (buffer only). IC50 values are reported in table 3.

TABLE 3 ddPCR assay on primary hepatocytes

Duplex numbering	IC50(μM)	% HSD17B13 knockdown
			D-2107	0.0112	-88.9134
D-2015	0.0112	-91.9705
			D-2016	0.0296	-87.2192
D-2014	0.0343	-80.4788

Example 4: screening of chemically modified HSD17B13 siRNA molecules in wild type rats

Sprague Dawley rats with body weights of 350-400gm at 9 to 10 weeks of age were obtained from Charles river laboratory (Charles River Laboratories) (Charles river laboratory Co., ltd. (Charles Rivers Laboratory, inc.), mass.). After acclimation, the animals were randomly grouped based on body weight. Each group included 6 rats and HSD17B13 siRNA was administered subcutaneously at 3 mg/kg body weight. The compounds were diluted in calcium and magnesium free phosphate buffer (Semerle Feier technologies (Thermo Fischer Scientific), 14190-136). At 30 days after siRNA treatment, animals were euthanized and livers were obtained. The newly isolated left liver leaf was immediately snap frozen in liquid nitrogen. 30-50mg of liver tissue was used to isolate RNA using a QIAcube HT instrument and the RNeasy 96QIAcube HT kit according to the manufacturer's protocol. 2-4ug of RNA was treated with DNase (Promega, M6101) without RQ1 RNase. Real-time qPCR was performed on 10ng of dnase digested RNA using the TaqMan RNA to CT 1 step method kit (applied biosystems (Applied Biosystems)) running on a Quant Studio real-time PCR machine. The expression was measured using the TaqMan probe of rat HSD17B13 (rn_ 01450039_m1,Invitrogen Taqman expression assay) and normalized to housekeeping gene HMBS (hydroxymethyl bile synthase Rn01421873_g1, invitrogen Taqman expression assay) expression. The relative fold change was calculated compared to the PBS group. Data are expressed as percent knockdown of siRNA treated groups relative to PBS. A total of 23 triggers were tested. The results are shown in Table 4. Negative values indicate elevated levels of HSD17B 13.

Table 4: percent silencing of day 30-siRNA HSD17B13 treated rats

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Claims

1. An RNAi construct comprising a sense strand and an antisense strand, wherein the antisense strand comprises a region having at least 15 contiguous nucleotides differing by no more than 3 nucleotides from the antisense sequence set forth in table 1 or table 2, and wherein the RNAi construct inhibits expression of 17β -hydroxysteroid dehydrogenase type 13 (HSD 17B 13).

2. The RNAi construct of claim 1, wherein the antisense strand comprises a region complementary to HSD17B13mRNA sequence.

3. The RNAi construct of any one of the preceding claims, wherein the sense strand comprises a region having at least 15 contiguous nucleotides differing by no more than 3 nucleotides from the antisense sequence set forth in table 1 or table 2.

4. The RNAi construct of any one of the above claims, wherein the sense strand comprises a sequence sufficiently complementary to the sequence of the antisense strand to form a duplex region of about 15 to about 30 base pairs in length.

5. The RNAi construct of claim 4, wherein the duplex region is about 17 to about 24 base pairs in length.

6. The RNAi construct of claim 4, wherein the duplex region is about 19 to about 21 base pairs in length.

7. The RNAi construct of claim 6, wherein the duplex region is 19 base pairs in length.

8. The RNAi construct of claim 6, wherein the duplex region is 20 base pairs in length.

9. The RNAi construct of claim 6, wherein the duplex region is 21 base pairs in length.

10. The RNAi construct of any one of claims 4-9, wherein the sense strand and the antisense strand are each about 15 to about 30 nucleotides in length.

11. The RNAi construct of claim 10, wherein the sense strand and the antisense strand are each about 19 to about 27 nucleotides in length.

12. The RNAi construct of claim 10, wherein the sense strand and the antisense strand are each about 21 to about 25 nucleotides in length.

13. The RNAi construct of claim 10, wherein the sense strand and the antisense strand are each about 21 to about 23 nucleotides in length.

14. The RNAi construct of any one of claims 1-13, wherein the RNAi construct comprises at least one blunt end.

15. The RNAi construct of any one of claims 1-13, wherein the RNAi construct comprises at least one nucleotide overhang of 1-4 unpaired nucleotides.

16. The RNAi construct of claim 15, wherein the nucleotide overhang has 2 unpaired nucleotides.

17. The RNAi construct of claim 15 or 16, wherein the RNAi construct comprises a nucleotide overhang at the 3 'end of the sense strand, the 3' end of the antisense strand, or both the sense and antisense strands.

18. The RNAi construct of any one of claims 15-17, wherein the nucleotide overhang comprises a 5'-UU-3' dinucleotide or a 5'-dTdT-3' dinucleotide.

19. The RNAi construct of any one of claims 1-18, wherein the RNAi construct comprises at least one modified nucleotide.

20. The RNAi construct of claim 19, wherein the modified nucleotide is a 2' -modified nucleotide.

21. The RNAi construct of claim 19, wherein the modified nucleotide is a 2 '-fluoro modified nucleotide, a 2' -O-methyl modified nucleotide, a 2 '-O-methoxyethyl modified nucleotide, a 2' -O-allyl modified nucleotide, a Bicyclic Nucleic Acid (BNA), a diol nucleic acid, an inverted base, or a combination thereof.

22. The RNAi construct of claim 21, wherein the modified nucleotide is a 2' -O-methyl modified nucleotide, a 2' -O-methoxyethyl modified nucleotide, a 2' -fluoro modified nucleotide, or a combination thereof.

23. The RNAi construct of claim 19, wherein all nucleotides in the sense strand and the antisense strand are modified nucleotides.

24. The RNAi construct of claim 23, wherein the modified nucleotides are 2 '-O-methyl modified nucleotides, 2' -fluoro modified nucleotides, or a combination thereof.

25. The RNAi construct of any one of claims 1-24, wherein the RNAi construct comprises at least one phosphorothioate internucleotide linkage.

26. The RNAi construct of claim 25, wherein the RNAi construct comprises two consecutive phosphorothioate internucleotide linkages at the 3' end of the antisense strand.

27. The RNAi construct of claim 25, wherein the RNAi construct comprises two consecutive phosphorothioate internucleotide linkages at the 3' and 5' ends of the antisense strand, and two consecutive phosphorothioate internucleotide linkages at the 5' end of the sense strand.

28. The RNAi construct of any one of claims 1-27, wherein the antisense strand comprises a sequence selected from the antisense sequences listed in table 1 or table 2.

29. The RNAi construct of claim 28, wherein the sense strand comprises a sequence selected from the sense sequences listed in table 1 or table 2.

30. The RNAi construct of any one of claims 1-29, wherein the RNAi construct is any one of the duplex compounds listed in any one of tables 1-2.

31. The RNAi construct of any one of claims 1-30, wherein the RNAi construct reduces HSD17B13 expression levels in liver cells after incubation with the RNAi construct compared to HSD17B13 expression levels in liver cells that have been incubated with a control RNAi construct.

32. The RNAi construct of claim 25, wherein the liver cell is a primary liver cell.

33. The RNAi construct of any one of claims 1-32, wherein the RNAi construct inhibits expression of HSD17B13 in primary hepatocytes with an IC50 of less than about 40 nM.

34. The RNAi construct of any one of claims 1-32, wherein the RNAi construct inhibits expression of HSD17B13 in primary hepatocytes with an IC50 of less than about 30 nM.

35. A pharmaceutical composition comprising the RNAi construct of any one of claims 1-34 and a pharmaceutically acceptable carrier, excipient, or diluent.

36. A method for reducing HSD17B13 expression in a patient in need thereof, the method comprising administering to the patient the RNAi construct of any one of claims 1-35.

37. The method of claim 36, wherein the expression level of HSD17B13 in hepatocytes is reduced in a patient following administration of the RNAi construct compared to the expression level of HSD17B13 in a patient not receiving the RNAi construct.